Sistema reprodutor masculino e

UNIVERSIDADE ESTADUAL PAULISTA – UNESP

CENTRO DE AQUICULTURA DA UNESP

Sistema reprodutor masculino e

espermiotaxonomia em Dromiidae: existe um só

padrão de produção de fluido seminal,

empacotamento do espermatozoide e

transferência espermática em caranguejos?

Maria Alice Garcia Bento

Jaboticabal, São Paulo

2018

UNIVERSIDADE ESTADUAL PAULISTA – UNESP

CENTRO DE AQUICULTURA DA UNESP

Sistema reprodutor masculino e espermiotaxonomia em

Dromiidae: existe um só padrão de produção de fluido

seminal, empacotamento do espermatozoide e

transferência espermática em caranguejos?

Maria Alice Garcia Bento

Orientador: Dr. Fernando José Zara

Coorientadora: Dra. Laura López Greco

Dissertação apresentada ao Programa de

Pós-graduação em Aquicultura do Centro

de Aquicultura da UNESP - CAUNESP,

como parte dos requisitos para obtenção do

título de Mestre em Aquicultura (Biologia

Aquática).

Jaboticabal, São Paulo

2018

Bento, Maria Alice Garcia

B478s Sistema reprodutor masculino e espermiotaxonomia em Dromiidae: existe um só padrão de produção de fluido seminal, empacotamento do espermatozoide e transferência espermática em caranguejos?/ Maria Alice Garcia Bento. – – Jaboticabal, 2018

139 p. : il. ; 29 cm

Dissertação (mestrado) - Universidade Estadual Paulista, Centro

de Aquicultura, 2018

Orientador: Fernando José Zara Coorientadora: Laura López Greco

Banca examinadora: Fernando Luis Medina Mantelatto, Marcos Domingos Siqueira Tavares

Bibliografia

1. Decapoda. 2. Transferência espermática. 3. Espermatozoides-

ultraestrutura. 4. Sistema reprodutor masculino. 5. Armazenamento dos espermatozoides. I. Título. II. Jaboticabal-Centro de Aquicultura.

CDU 595.84:591.16

Caunesp

“Nunca deixe que lhe digam que não vale a pena

acreditar no sonho que se tem,

ou que seus planos nunca vão dar certo

ou que você nunca vai ser alguém”

Flávio Venturini / Renato Russo

Dedico este trabalho à minha família e

em especial à minha mãe Maria

do Carmo, meu pai Antônio Bento e a

minha irmã Beatriz, por todo o amor e

confiança.

AGRADECIMENTOS

À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

pelo suporte financeiro concedido durante todo o desenvolvimento deste estudo,

por meio da bolsa de Iniciação Científica (Processo 2014/21294-5) vinculada ao

projeto BIOTA- FAPESP (Processo 2010/50188-8) e pela bolsa de mestrado

(Processo 2016/10394-4). Agradeço ainda á Coordenação de Aperfeiçoamento de

Nível Superior - CAPES - Ciências do Mar (CIMAR) II (Processo 1889/2014-

23038.004309/2014-51) pelos auxílios concedidos ao Prof. Dr. Fernando José

Zara, nos quais auxiliaram no custeio dos reagentes utilizados durante a pesquisa

e nas atividades de campo.

À Deus, por ter me acompanhado em todas as etapas da minha vida até

este momento e especialmente durante a execução deste estudo. Obrigada por

todas as oportunidades, proteção, amor, confiança e força transmitida para me

manter firme e seguir sempre em frente. Ao TLC e aos grandes amigos que me

apresentou.

Ao meu orientador Prof. Dr. Fernando José Zara, por todo tempo que

dedicou ao meu crescimento, por todo auxílio, atenção, paciência, amizade,

confiança e incentivo. Agradeço ainda por sempre me inspirar a buscar novos

conhecimentos e por toda empolgação com cada parte deste estudo conquistada.

Este seu diferencial me fez buscar ser diferente.

À minha coorientadora Prof. Dra. Laura López Greco por todo auxílio,

correções, paciência, amizade e ensinamentos durante a execução deste trabalho

e minha estadia em Buenos Aires – Argentina, para a participação no curso de

“Morfología funcional de la reproducción y el crecimiento de crustáceos

decápodos: aspectos teóricos y aplicados. Saiba que graças ao seu apoio, além

de adquirir novos conhecimentos sobre crustáceos, obtive um crescimento

pessoal imensurável.

Ao Prof. Dr. Fernando L. Mantelatto e à Dra. Ivana Miranda Trettin e ao

Laboratório de Bioecologia e Sistemática de Crustáceos (LBSC) do Departamento

de Biologia da Faculdade de Filosofia Ciências e Letras de Ribeirão Preto

(FFCLRP), por todo auxílio e suporte concedidos na produção do primeiro

capítulo desta dissertação.

À Marcia Fiorese Mataqueiro, técnica e grande amiga que encontrei no

Laboratório de Morfologia de Invertebrados da Unesp de Jaboticabal, que me

auxiliou e ensinou todas as técnicas laboratoriais com amor e paciência. Obrigada

por todos os conselhos, puxões de orelha e amizade. Você não é 10, é 1000.

Ao grande parceiro e amigo Djalma Rosa, vulgo passarinho, pela coleta de

todos os animais utilizados neste estudo e por toda a ajuda de sempre.

Aos amigos, companheiros e ex-companheiros do Laboratório de

Morfologia de Invertebrados: Tavani, Jean, Camila, Fernanda, Bárbara,

Guilherme, Lucas, Gisele, Timóteo e Leo. Por todos os conhecimentos, trabalhos,

esforços, alegrias e comilanças compartilhadas. Em especial a Camila e

Fernanda que me deram forças e ajuda nas correções finais.

À Milena Wolf, pela amizade, conselhos e companhia. Ao pessoal do Biota

Fapesp e todos os laboratórios associados a este projeto.

À técnica Claudia Maria e ao Laboratório de Microscopia eletrônica da

Unesp de Jaboticabal por todo suporte e apoio concedidos em muitas etapas

deste estudo.

Ao Prof. Davi Rossato e a Ivana Trettin, pelas sugestões e correções

efetuadas na banca de qualificação.

À Marcia Macri por todas as vezes que me auxiliou e me ajudou com as

papeladas.

Aos funcionários do Departamento de Biologia Aplicada a Agropecuária.

Ao Caunesp, David, professores e todos os funcionários desta instituição,

pela oportunidade do mestrado, pelos auxílios, disciplinas e por tudo o que

aprendi durante o mestrado.

Às minhas amigas Luana Trevisanuto e Tatiane Mattos por toda amizade e

apoio de sempre.

A todos os meus familiares e ao meu Pai Antonio Bento, por todo amor,

confiança, incentivo e apoio.

À minha vózinha, por todas as rezas, pensamentos positivos e amor que

me dedicou. E ao meu avô, que não está entre nós, mas com certeza está no céu

radiante ao ver minha conquista.

Ao meu amor Gabriel Yuri, pelo amor, paciência, cumplicidade, amizade,

ajuda, incentivos e por tudo o que vivemos. Você é muito importante. Obrigada

por me dar força em todas as situações. Te amo.

À Carla, Flavio, Nathalie e Lionaldo, pelo apoio, carinho e acolhida de

sempre. Vocês são pessoas muito importantes e especiais.

Ao Carlão, por dar apoio a minha mãe e por nos tratar sempre muito bem.

Em especial à minha mãe Maria do Carmo e minha irmã Beatriz, pela

convivência, carinho, apoio, paciência, cumplicidade, confiança, incentivo,

conselhos e muito amor. Obrigada por serem meus pilares e por não me deixarem

fraquejar. Amo vocês demais.

Muito obrigada!!

APOIO FINANCEIRO

FAPESP, Bolsa de Iniciação Científica, Processo nº 2014/21294-5

FAPESP, Bolsa de Mestrado, Processo nº 2016/10394-4

BIOTA FAPESP, Processos nº 2010/50188-8

Ciências do Mar (CIMAR) II, Processo nº 1889/2014-23038.004309/2014-51

SUMÁRIO

Resumo...........................................................................................................................12

Abstract..........................................................................................................................13

Introdução geral............................................................................................................14

Referências……………………………………………………………………….....…20

Capítulo I. Sperm ultrastructure of four crab species of the family Dromiidae (Crustacea:

Brachyura): addition data to Hypoconchinae and Dromiinae subfamilies……………..24

Abstract………………………………………………………………...…………26

Introduction…………………………………………………………………….....27

Materials and Methods……………………………………………………….......28

Animal samples…………………………………………………………..…28

Sperm ultrastructure.....................................................................................29

Results......................................................................................................................30

General diagram............................................................................................30

Species spermatozoa ultrastructure..............................................................30

Discussion……………………………………………………………………….….34

Acknowledgments……………………………………………………………........38

References………………………………………………………………………….38

List of legends................................……………………………………………...…42

List of figures and table…………………………………………………………...47

Capítulo II. Seminal fluid production and sperm transfer in dromiids: new insights into

the evolution of crab reproduction……………………………………………………..54

Abstract…………………………………………………………………………...56

Introduction………………………………………………………………………57

Materials and Methods……………………………………………………..……59

Results………………………………………………………………………..…...61

Gross anatomy………………………………………….……………….....61

Histology and Histochemistry…………………………………………….62

Testis……………………………………………………………………..62

Vas deferens………………………………………………………….…..63

Ultrastructure of the vas defeens…………………………………...…....65

Mobile penial tube and gonopod…………………………………..….....66

Differential Interference Contrast Phase Microscope (DIC)………….....67

“Sperm plug” and first feale pleopod…………………………......…......67

Discussion……………………………………………………………………........67

Acknowledgments…………………………………………………………….......75

References………………………………………………………………………....75

List of legends….……………………………………………………....................80

List of legends and table........................................................................................88

Capítulo III. Comportamento de cópula e armazenamento espermático no caranguejo

Hypoconcha parasitica (Podotremata: Dromiidae).............................................100

Resumo………………………………………………………………………........102

Introdução…………………………………………………………………....…...103

Material e Métodos………………………………………………………............105

Animais……………………………………………………………........…...105

Comportamento reprodutivo.......................................................................105

Análise da espermateca e ovários.................................................................106

Resultados................................................................................................................107

Comportamento reprodutivo.......................................................................107

Espermateca e estruturas associadas...........................................................109

Discussão.................................................................................................................112

Agradecimentos......................................................................................................117

Referências..............................................................................................................118

Lista de legendas....................................................................................................122

Lista de figuras.......................................................................................................126

Conclusão geral......................................................................................................133

Mestranda Maria Alice Garcia Bento Orientador - Fernando José Zara

12 Caunesp

RESUMO

Os caranguejos da família Dromiidae estão incluídos na seção Podotremata Guinot,

1977 e são pouco conhecidos do ponto de vista histo-morfológico e ultraestrutural.

Na presente dissertação, apresentamos a ultraestrutura dos espermatozoides e os

padrões anátomo-histológicos do sistema reprodutor dos dromiídeos que ocorrem no

litoral brasileiro. Adicionalmente, o comportamento de cópula e a morfologia da

espermateca em Hypoconcha parasitica foram estudados, com o intuito de levantar

pistas sobre os mecanismos de fertilização em Dromiidae. As amostras foram

processadas segundo as rotinas para histologia e histoquímica, microscopia,

eletrônica de transmissão e varredura. As análises ultraestruturais do

espermatozoide indicam que não existe um padrão distinto típico para os gametas

entre todos os Dromioidea. Adicionalmente, apenas caracteres específicos foram

observados, como a presença do flange esférico na zona eletronlúcida anterolateral

e a zona acrossomal externa granular em H. parasitica, a ausência da zona

acrossomal raiada e a presença de resquícios de flange em Moreiradromia

antillensis e diferentes morfologias das câmaras perforatoriais entre todas as

espécies estudadas. Apenas quando os caracteres da ultraestrutura dos

espermatozoides dos Dromioidea foram comparados com Homolidae, Latreillidae e

Raninidae, notaram-se características claras entre estas famílias. A produção do

fluido seminal e o empacotamento dos espermatozoides no vaso deferente dos

Dromiidae são totalmente distintos dos Eubrachyura. Hypoconcha parasitica,

Hypoconcha arcuata, M. antillensis e Dromia erythropus não possuem

espermatóforos, mas sim a presença de um cordão espermático interno envolto por

secreções, os quais são mais similares aos Astacidae. O comportamento de cópula

em H. parasitica é simples, sem corte e as fêmeas copulam em muda e intermuda. O

armazenamento do material seminal na espermateca é distinto de Eubrachyura. A

organização morfo-histológica da espermateca indica que a liberação dos

espermatozoides para a fertilização ocorre por meio de contrações musculares da

parede cuticular, com a participação do pleópodo 1 na movimentação dos ovócitos e

espermatozoides, auxiliando o contato dos gametas.

PALAVRAS-CHAVE: Decapoda, transferência espermática, ultraestrutura; sistema

reprodutor masculino, armazenamento dos espermatozoides.


13 Caunesp

ABSTRACT

The Dromiidae crabs are included in the section Podotremata Guinot, 1977 and

are poorly known from histo-morphological and ultrastructural point of view. This

dissertation shows the sperm ultrastructure and the anatomo-histological patterns

of the reproductive system to the Brazilian dromiid species. In addition, we studied

the mating behavior and spermathecal morphology in Hypoconcha parasitica to

elucidate some points of the fertilization in dromiid crabs. The samples were

processed according to the routine for histology and histochemistry and also, to

transmission and scanning electron microscopy. There are no distinct

ultrastructural characters to sperm ultrastructure among Dromioidea. Additionally,

only specific characters were observed for studied Dromiidae as the presence of

the spherical flange in the anterolateral eletron-lucid zone and the granular

acrosomal outer zone founded in H. parasitica, absence of the acrosome ray zone

and presence of the flange remnants in Moreiradromia antillensis. The

perforatorial chambers show specific morphology among all species studied. The

Dromiidae spermatozoa ultrastructure were not consistent to separate Dromiidae

from the other Dromioidea. The ultrastructural differences were only found when

compared Dromiodea with other Podotremata as Homolidae, Latreillidae e

Raninidae. The seminal fluid production and spermatozoa package in the vas

deferens of Dromiidae are totally different from Eubrachyura. In H. parasitica,

Hypoconcha arcuata, M. antillensis and Dromia erythropus, the spermatophores

are absent, and they show only a long internal spermatic cord surrounded by

secretions in vas deferens, which is similar to the species of Astacidae. The

mating behavior is simple without courtship and the females copulated in both soft

and hard during moulting condition. The storage of seminal material inside the

spermatheca is distinct from Eubrachyura. The morpho-histological organization of

the spermathecae indicates that the process of sperm release for fertilization

occurs through muscular contractions of its wall, and the pleopod 1 may promote

the oocytes and spermatozoa movement to ensure the fertilization.

KEY-WORDS: Decapoda, sperm transfer; histology, ultrastructure, male

reproductive system, sperm storage.


14 Caunesp

INTRODUÇÃO GERAL

A família Dromiidae (De Haan, 1883) pertence à infraordem Brachyura,

incluída na seção Podotremata Guinot, 1977, na qual agrupa espécies em que o

sistema reprodutor masculino abre-se na coxa do quinto pereópodo e as fêmeas

mostram duas aberturas: uma do oviduto (gonóporo), por onde os ovócitos são

liberados, localizada na coxa do terceiro pereópodo e a espermateca, cujo é um

canal estendido que se abre em diferentes esternitos, dependendo das famílias

e subfamílias (Guinot & Tavares, 2001; Guinot & Quenette, 2005; Guinot et al.,

2013). Com aproximadamente 120 espécies taxonomicamente válidas, os

representantes de Dromiidae encontram-se subdivididos nas subfamílias

Dromiinae De Haan, 1833, Hypoconchinae Guinot & Tavares, 2003 e

Sphaerodromiinae Guinot & Tavares 2003, as quais diferem-se principalmente

pela organização do esterno torácico, abdômen, coxa do quinto pereiópodo e

pênis nos machos, além das estruturas do esterno torácico 7/8 nas fêmeas

(Guinot & Tavares, 2001; 2003; Ng et al., 2008). No Brasil são encontrados

somente representantes das subfamílias Dromiinae e Hypoconchinae,

representados pelas espécies Dromia erythropus (George Edwards, 1771),

Moreiradromia antillensis (Stimpson, 1858), Hypoconcha parasitica (Linnaeus,

1763) e H. arcuata Stimpson, 1858 (Melo, 1996; Ng et al., 2008). Estes

dromiídeos são comumente conhecidos pela associação com conchas de

bivalves, esponjas ou ascídias, as quais são posicionadas dorsalmente ao

animal, com auxílio dos dois últimos pares de patas, dobradas na região sub-

dorsal da carapaça (Mclay, 1993; Melo, 1996; Guinot et al., 2013).

De uma maneira geral, o sistema reprodutor em Brachyura é um órgão

bilateral em forma de letra “H”, constituído pelo par de testículos, localizado em

ambas às margens superiores do cefalotórax, os quais são contínuos ao vaso

deferente, estendendo-se longitudinalmente sobre o hepatopâncreas ou órgão

perigástrico de acordo com Cervellione et al. (2017), abaixo do coração,

terminando na região posterior do corpo (Krol et al., 1992). O testículo pode ser

classificado como do tipo lobular ou tubular (para revisão ver Nagao &

Munehara, 2003). O vaso deferente é anatomicamente dividido em três regiões:

anterior (AVD), média (MVD) e posterior (PVD) (Krol et al., 1992). A AVD tem a

função de produzir as secreções que levarão a formação dos espermatóforos


15 Caunesp

(Simeó et al., 2009; Nicolau et al., 2012; Zara et al., 2012). Por sua vez, a MVD e

PVD são responsáveis por armazenar os espermatóforos e produzir a maior

parte do fluido seminal (Krol et al., 1992; Simeó et al., 2009; Zara et al., 2012).

Em grande parte dos braquiúros, os espermatozoides são empacotados em

espermatóforos do tipo coenospérmicos, nos quais vários espermatozoides

encontram-se agrupados e delimitados por uma só parede do espermatóforo (El-

Sherief, 1991; Jamieson, 1994; Guinot, 1997; Anilkumar et al., 1999). Porém, em

algumas espécies de caranguejos de água doce e, em ao menos uma espécie

marinha, cada espermatóforo apresenta um só espermatozoide, sendo

denominado como cleistoespérmico (Guinot, 1997; Klaus et al., 2009; Tiseo et

al., 2014; Tiseo et al., 2017). A ausência de espermatóforos é um evento raro em

Brachyura e foi somente observada em alguns caranguejos de água doce (Klaus

et al., 2009).

A descrição do sistema reprodutor masculino em espécies primitivas de

Brachyura é bastante escassa, sendo que apenas o Raninidae Ranina ranina

(Linnaeus, 1758) e o Dromiidae Dromia personata (Linnaeus, 1758) foram

estudados sob este aspecto (Hartnoll, 1975; Subramoniam, 1993; Minagawa et

al., 1994). O testículo de R. ranina foi classificado histologicamente como do tipo

lobular e o vaso deferente mostrou três regiões distintas, i.e. AVD, MVD e PVD,

sendo os espermatozoides, nesta estrutura, envolto por cápsula ou parede,

positiva ao corante ácido periódico de Schiff (PAS), o qual evidencia a presença

de polissacarídeos neutros, sendo denominada de espermatóforo (Minagawa,

1993; Minagawa et al.,1994). Adicionalmente, estes autores não discutem a

evolução da massa espermática, o significado da cápsula ou se esta estrutura

pode ser ou não considerada um espermatóforo verdadeiro. Similar ao padrão

encontrado em R. ranina, o vaso deferente de Dromia personata (Linnaeus,

1758) também foi dividido em três diferentes regiões, porém ao invés de

relacionada à anatomia, a divisão foi baseada nos diferentes tipos de secreções

e constituição da parede do vaso deferente (Hartnoll,1975; Minagawa et al.,

1994). A estruturação das secreções no vaso deste Dromiidae pareceu ser mais

similar ao padrão encontrado para espécies de Astacidea, uma vez que não

mostraram parede típica dos espermatóforos, mas sim diferentes tipos de

secreções sendo aderidas em camadas (Hartnoll, 1975; Subramoniam 1993).


16 Caunesp

Desta forma, nota-se que o conhecimento do sistema reprodutor masculino em

Podotremata carece de maiores aprofundamentos.

Os espermatozoides de Decapoda são imóveis e aflagelados, podendo ser

classificados morfologicamente em duas categorias: uniestelado, encontrados

em Dendrobranchiata e Caridea; ou multiestelado encontrados em Brachyura,

Anomura e Pleocyemata (Jamieson & Tudge, 2000; Braga et al., 2013; Camargo

et al., 2016; Tiseo et al., 2017). Em Brachyura, o espermatozoide é constituído

em geral pelo núcleo, vesícula acrossomal, a qual porta o opérculo e uma

estrutura perfuradora, a câmara perforatorial. A vesícula acrossomal encontra-se

em um dos pólos do espermatozoide, o oposto ao núcleo. Esta estrutura é

marcada por diferentes zonas ou camadas acrossomais (Jamieson, 1994;

Jamieson & Tudge, 2000, Tudge, 2009). No ápice da vesícula acrossomal

encontra-se o opérculo eletrondenso, a qual pode ser ou não perfurado. A

câmara perforatorial (tubo perfurador) encontra-se na região central do

acrossoma, a qual pode apresentar ampla variação de forma (Jamieson &

Tudge, 2000; Tudge, 2009). O núcleo é preenchido por cromatina variando de

granular a fibrosa e pode apresentar braços radiais com ou sem microtúbulos

(Hinsch, 1986; Jamieson, 1994; Benetti et al., 2008; Klaus et al., 2009; Tudge,

2009). Em relação à ultraestrutura dos espermatozoides em Dromiidae, apenas

os espermatozoides do Sphaerodromiinae Sphaerodromia lamellata Crosnier,

1994 e dos Dromiinae Stimdromia lateralis (Gray, 1831) e Dromidiopsis edwardsi

Rathbun, 1919 foram estudados sob este ponto de vista (Jamieson et al., 1993

a,b,c; Jamieson, 1994; Guinot et al., 1998; Jamieson & Tudge, 2000). De uma

maneira geral, nestas espécies o espermatozoide apresenta acrossomo do tipo

discóide com câmara perforatorial bilateralmente capitada e, a partir desta,

ocorrem quatro zonas horizontais concêntricas: zona acrossomal interna, zona

acrossomal raiada, zona acrossomal mediana ou eletronlúcida anterolateral e

zona acrossomal externa (Jamieson et al., 1993 a,b,c; Jamieson, 1994; Guinot et

al., 1998; Jamieson & Tudge, 2000). O opérculo apical mostra-se perfurado e

centralmente a perfuração existe uma protuberância apical (Jamieson, 1994). O

núcleo é preenchido por cromatina fibrosa, podendo ou não apresentar braços

radiais (Jamieson et al., 1993 a,b,c; Jamieson, 1994; Jamieson et al., 1995;

Guinot et al., 1998; Jamieson & Tudge, 2000). Apesar de existirem trabalhos

com Dromiidae, as inclusões das espécies da fauna brasileira contribuirão para


17 Caunesp

uma maior elucidação filogenética das espécies que pode ser concatenada à

molecular, existente na literatura. Adicionalmente, em Dromiidae uma das

subfamílias, Hypoconchinae, tem o espermatozoide totalmente desconhecido do

ponto de vista ultraestrutural. Desta maneira, conciliando os resultados deste

estudo com a molecular, será possível aumentar o poder de resolução nas

relações de parentesco.

A transferência espermática esta diretamente relacionada ao momento em

que as fêmeas tornam-se receptivas aos machos, podendo realizar a atração

destes por meio de, por exemplo, feromônios liberados na urina (mais frequente)

ou estímulo tátil, visual ou auditivo (menos frequente) (Hartnoll, 1969, Kennedy &

Cronin, 2007). Em relação à receptividade das fêmeas, aquelas capazes de

copular durante a muda, (exoesqueleto mole) encontram-se receptivas aos

machos apenas em curtos períodos, enquanto que as fêmeas capazes de

copular em intermuda (exoesqueleto rígido) encontram-se receptivas

continuamente (Diesel, 1991; Mclay & López Greco, 2011). Em Dromiidae, o

acasalamento em Dromia personata Linnaeus, 1758 pode ocorrer pós muda

(muda pré-ovígera), quando o exoesqueleto encontra-se mole e ao menos em

um caso, a cópula ocorreu enquanto a fêmea estava na condição de intermuda,

com o exoesqueleto rígido (Hartnoll, 1975). Apesar de mostrar estes resultados,

o autor não discute no que implicaria o acasalamento ocorrer na condição de

muda ou intermuda, tampouco descreve onde os gonópodos são inseridos para

a transferência espermática. Adicionalmente, os repertórios comportamentais

durante a transferência espermática ou sua relação com o ciclo de muda

também são pouco abordados para D. personata e outras espécies de

Podotremata (Hartnoll, 1975; Guinot et al.,2013). Desta maneira, nota-se uma

lacuna a respeito da transferência espermática em Podotremata, visto que este

tema nunca foi abordado na íntegra em espécies deste grupo, sendo este de

grande importância para o entendimento de estruturas morfológicas, com ênfase

na função da espermateca, nos modos reprodutivos e a evolução dos órgãos de

armazenamento do fluido seminal e fêmeas dos caranguejos.

Nos Eubrachyura, as fêmeas possuem uma estrutura responsável em

armazenar espermatozoides após a cópula, com origem ecto-mesoderme

(Diesel, 1991; Guinot & Quenette, 2005; Guinot et al., 2013; Mclay & Becker,

2015). Esta estrutura já foi denominada como espermateca (espermathecae)


18 Caunesp

(Hartnoll 1969; Beninguer et al., 1988; López Greco et al., 1999; Rotllant et al.,

2007; Sant´Anna et al., 2007). Porém, o termo mais apropriado para esta

estrutura de origem mista é receptáculo seminal (Guinot & Ouenette, 2005;

López Greco et al., 2009; Zara et al., 2014). No receptáculo seminal, o oviduto

tem contato direto com a região de armazenamento dos espermatozoides e este

contato pode ser dorsal ou ventral, influenciando decisivamente na fertilização

dos ovócitos. Assim, este órgão pode favorecer o primeiro ou último macho que

realizou a cópula, dependendo do local de abertura do oviduto (Diesel, 1991;

Antunes et al., 2016). O termo espermateca ficou restrito a estruturas de origem

exclusivamente ectodérmica, observado em Podotremada (Tavares & Secretan,

1993; Guinot & Tavares, 2001; Guinot & Ouenette, 2005). Neste grupo, os

machos possuem o par de pênis, gonóporos, gonópodos localizados na coxa do

quinto par de pereópodos, e gonópodos, com diferentes morfologias e funções

para cada família e subfamília (para revisão ver Guinot & Tavares, 2003; Guinot

& Quenette, 2005; Guinot et al., 2013). Por sua vez, as fêmeas mostram duas

aberturas: o par de gonóporos, localizados na coxa do terceiro pereópodo, cuja

função é externalizar os ovócitos e o par de espermatecas. A espermateca é

uma depressão no esterno, cuja abertura é alongada em forma de fenda, sem

ligação interna com o oviduto (Guinot & Tavares, 2001; Guinot & Tavares, 2003;

Guinot & Quenette, 2005; Guinot et al., 2013). Como a espermateca não contém

conexão interna com o oviduto, acredita-se que os gametas masculinos

precisam ser deslocados desta estrutura quitinosa até a região externa do corpo

da fêmea para que a fertilização ocorra (Mclay & López Greco, 2011). Porém, o

mecanismo funcional desta estrutura ainda continua sendo um mistério, sendo

que em Homolidae foi sugerido que a ação de músculos na abertura

espermatecal contribuem para a liberação dos espermatozoides (Becker &

Scholtz, 2017).

Algumas espécies produzem o “plug espermático”, o qual é formado por

secreções seminais dos machos. O plug pode se estender desde o receptáculo

seminal até a região externa da vagina, ou ocupar somente a vagina (plug

externo) ou formar uma massa semelhante à cera no interior do receptáculo

seminal (plug interno) (Hartnol, 1968, 1969). Tais plugs parecem ser cruciais

para o favorecimento de um único macho durante a cópula, pois a presença

desta estrutura impossibilita ou dificulta o recebimento de material genético


19 Caunesp

adicional proveniente de outros machos (Hartnoll 1968, 1969; Zara et al., 2012;

Nascimento & Zara, 2013). A presença de plug é frequentemente atribuída às

espécies que acasalam com exoesqueleto ou carapaça mole, como por

exemplo, Portunidae (Hartnoll, 1969; Johnson, 1980; Zara et al., 2012;

Nascimento & Zara, 2013; Guinot et al., 2013; Zara et al., 2014). Além disso,

estas espécies podem apresentar o comportamento de guarda pré e pós

copulatória, nos quais os machos permanecem em “abraço” com a fêmea antes,

durante e após a muda, a fim de impedir que outros machos tentem copular com

a fêmea (Hartnoll, 1969; Zara et al., 2012). Em Podotremata, estudos com o

comportamento e transferência espermática são escassos, sendo que em R.

ranina, observou-se a presença de pequenos espermatóforos depositados junto

a espermateca após a cópula (Minagawa, 1993). Este mesmo autor propôs que

parte do espermatóforo estavam internalizados na espermateca, porém não

houve a devida comprovação por dissecção. No tocante a Dromiidae, o

conhecimento é restrito ao que foi descrito para Dromia personata (Hartnoll,

1975). Segundo o autor, no final da transferência espermática, nota-se material

enrijecido ou “plug espermático” sobre a abertura da espermateca (Hartnoll,

1975). Apesar de acreditarem que Hypoconchinae não apresenta “plug

espermático”, a presença deste “plug” foi observada para também nos Dromiinae

Lauridromia intermedia (Laurie, 1906), Austrodromidia octodentata (Haswell,

1882) e Pseudodromia latens Stimpson, 1858 (Guinot & Tavares, 2003; Guinot &

Quenette, 2005; Guinot et al., 2013). Entretanto, este assunto permanece ainda

a ser aprofundado.

Assim, nesta dissertação são apresentados três capítulos em formato de

artigos científicos. O primeiro trata- se da descrição ultraestrutural dos

espermatozoides de Dromia erythropus, Moreiradromia antillensis, Hypoconcha

arcuata e H. parasitica, com objetivo de relacionar os caracteres espermáticos

entre os dromiídeos e outras famílias de Podotremata, buscando uma melhor

compreensão sobre as relações filogenéticas deste grupo. Desta maneira, este

capítulo produz um embasamento sobre o parentesco entre os Dromiidae para a

elucidação do anatomo-histologia do sistema reprodutor desta família. O

segundo descreve a morfologia do sistema reprodutor masculino dos Dromiidae

encontradas no Brasil, com o intuito de verificar se este grupo de caranguejos

apresenta o mesmo padrão usualmente descrito para o Eubrachyura, com a


20 Caunesp

presença de espermatóforos e características químicas do fluido seminal. O

último capítulo utiliza a espécie H. parasitica para elucidar aspectos do

comportamento de cópula e da morfologia da espermateca em Dromiidae, com o

intuito de levantar pistas sobre os mecanismos de fertilização neste grupo de

caranguejos primitivos.

REFERÊNCIAS

ANILKUMAR, G.; SUDHA, K.; SUBRAMONIAM, T. 1999. Spermatophore Transfer and

Sperm Structure in the Brachyuran Crab Metopograpsus messor (Decapoda:

Grapsidae). Journal of Crustacean Biology, 19:361-370.

ANTUNES, M., ZARA, F. J.; LÓPEZ GRECO, L. S.; NEGREIROS‐FRANSOZO, M. L.

2016. Morphological analysis of the female reproductive system of Stenorhynchus

seticornis (Brachyura: Inachoididae) and comparisons with other Majoidea.

Invertebrate Biology, 135: 75-86.

BECKER, C.; SCHOLTZ, G. 2017. Phylogenetic implications of sperm storage in

Podotremata: Histology and 3D‐reconstructions of spermathecae and gonopores in

female carrier crabs (Decapoda: Brachyura: Homoloidea). Journal of Morphology,

278: 89-105.

BENETTI, A. S.; SANTOS, D. C.; NEGREIROS-FRANSOZO, M. L.; SCELZO, M. A.

2008. Spermatozoal ultrastructure in three species of the genus Uca Leach, 1814

(Crustacea, Brachyura, Ocypodidae).Micron, 39:337-343.

BENINGER, P. G.; ELNER, R. W.; FOYLE, T. P.; ODENSE, P. H. 1988. Functional

anatomy of the male reproductive system and the female spermatheca in the snow

crab Chionoecetes opilio (O. Fabricius) (Decapoda: Majidae) and a hypothesis for

fertilization. Journal of Crustacean Biology, 322-332.

BRAGA, A. L.; NAKAYAMA, C. L.; POERSCH, L.; WASIELESKY, W. 2013. Unistellate

spermatozoa of decapods: comparative evaluation and evolution of the morphology.

Zoomorphology, 132:261–284.

CAMARGO, T. R.; ROSSI, N.; CASTILHO, A. L.; COSTA, R. C.; MANTELATTO, F. L.;

ZARA, F. J. 2016. Integrative analysis of sperm ultrastructure and molecular

genetics supports the phylogenetic positioning of the sympatric rock shrimps

Sicyonia dorsalis and Sicyonia typica (Decapoda, Sicyoniidae). Zoomorphology,

135, 67-81.

CERVELLIONE, F.; MCGURK, C.; VAN DEN BROECK, W. 2017. “Perigastric organ”: a

replacement name for the “hepatopancreas” of Decapoda. The Journal of

Crustacean Biology, 37: 353-355.

DIESEL, R. 1991. Sperm competition and the evolution of mating behavior in Brachyura,

with special reference to spider crabs (Decapoda, Majidae), In: J. MARTIN & R.

BAUER, Crustacean Sexual Biology, Columbia University Press, New York, 145-

163.

EL-SHERIEF, S. S. 1991. Fine Structure of the Sperm and Spermatophores of Portunus

pelagicus (L.)(Decapoda, Brachyura). Crustaceana, 61:271-279.


21 Caunesp

GUINOT D. 1997. Propositions pour une nouvelle classification des Crustacés

Décapodes Brachyoures. Comptes rendus hebdomadaires des Séances

del’Académie des Sciences, 285:1049-1052.

GUINOT, D.; JAMIESON, B. G. M.; FORGES, B. R. 1998. Comparative spermatozoal

ultrastructure of the three Dromiacean families exemplified by Homolodromia kai

(Homolodromiidae), Sphaerodromia lamellata (Dromiidae) and Dynomene tanensis

(Dynomenidae)(Podotremata: Brachyura). Journal of Crustacean Biology, 18: 78-94.

GUINOT, D.; QUENETTE, G. 2005. The spermatheca in Podotreme crabs (Crustacea,

Decapoda, Brachyura, Podotremata) and its phylogenetic implication. Zoosystema,

27:267-342.

GUINOT, D.; TAVARES, M. 2001. Une nouvelle famille de crabes du Crétacé, et la

notion de Podotremata Guinot, 1977 (Crustacea, Decapoda, Brachyura).

Zoosystema, 23:507–546.

GUINOT, D.; TAVARES, M. 2003. A new subfamilial arrangement for the Dromiidae de

Haan, 1833, with diagnoses and descriptions of new genera and species.

Zoosystema, 25:43-129.

GUINOT, D.; TAVARES, M.; CASTRO, P. 2013. Significance of the sexual openings and

supplementary structures on the phylogeny of brachyuran crabs (Crustacea,

Decapoda, Brachyura), with new nomina for higher-ranked podotreme taxa.

Zootaxa, 3665:1- 414.

HARTNOLL, R. G. 1968. Morphology of the genital ducts in female crabs. Journal of the

Linnean Society of London, Zoology, 47: 279-300.

HARTNOLL, R. G. 1969. Mating in the Brachyura. Crustaceana, 16:161-181.

HARTNOLL, R. G. 1975. Copulatory structure and function in the Dromiacea, and their

bearing on the evolution of the Brachyura. Publicationes Statione zoologico Napoli,

39:657-676.

HINSCH, G. W. 1986. A comparison of sperm morphologies, transfer and sperm mass

storage between two species of crab, Ovalipes ocellatus and Libinia emarginata.

International Journal of Invertebrate Reproduction and Development, 10:79-87.

JAMIESON, B. G. M. 1994. Phylogeny of the Brachyura with Particular Reference to the

Podotremata: Evidence from a Review of Spermatozoal Ultrastructure (Crustacea,

Decapoda). Philosophical Transactions: Biological Sciences, 345:373-393.

JAMIESON, B. G. M.; GUINOT, D.; FORGES, B. R. 1993c. Spermatozoal ultrastructure

in four genera of Homolidae (crustacea, decapoda): Exemplified by Homologenus

sp., Latreillopsis sp., Homolomannia sibogae and Paromolosis boasi. Helgoländer

Meeresuntersuchungen, 47:323-334.

JAMIESON, B. G. M.; GUINOT, D.; FORGES, B. R. 1993b. The ultrastructure of the

spermatozoon of Paradynomene tuberculata Sakai, 1963 (Crustacea, Brachyura,

Dynomenidae): synapomorphies with dromiid sperm. Helgolander

Meeresuntersuchungen, 47:311-322.

JAMIESON, B. G. M., GUINOT, D.; FORGES, B. R. 1995. Phylogeny of the Brachyura

(Crustacea, Decapoda): evidence from spermatozoal ultrastructure. In: Jamieson, B.

G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and

Taxonomy. Muséum.National d’ Histoire Naturelle, 166:265-283.

JAMIESON, B. G. M.; TUDGE, C. C.; SCHELTINGA, D. M. 1993a.The ultrastructure of

the Spermatozoon of Dromidiopsis edwarsi Rathbun, 1919 (Crustacea:Brachyura:

Dromiidae): Confirmation of a Dromiid Sperm Type. Australian Journal of Zoology,

41:537-48.


22 Caunesp

JAMIESON, B.G.M.; TUDGE, C. C. 2000. 1. Crustacea-Decapoda. In: Jamieson, B.G.M.

(Ed.), Progress in Male Gamete Ultrastructure and Phylogeny, 1-95.

JOHNSON, P. T. (ed.). 1980. Histology of the Blue Crab, Callinectes sapidus: A Model

for the Decapoda. Praeger, New York.

KENNEDY, V. S.; CRONIN, L. E. (Eds.). 2007. The blue crab: Callinectes sapidus.

Maryland Sea Grant College University of Maryland.

KLAUS, S.; SCHUBART, C. D.; BRANDIS, D. 2009. Ultrastructure of Spermatozoa and

Spermatophores of Old World Freshwater Crabs (Brachyura: Potamoidea:

Gecarcinucidae, Potamidae, and Potamonautidae). Journal of Morphology, 270:175-

193.

KROL, R. M.; HAWKINS, W. E.; OVERSTREET, R. M. 1992. Reproductive components,

pp. 295-343. In: Harrison, F. W.; Humes, A. G. (eds.), Microscopic Anatomy of

Invertebrates. Vol. 10: Decapod Crustacea, Wiley-Liss Inc., New York.

LÓPEZ GRECO, L. S.; FRANSOZO, V.; NEGREIROS-FRANSOZO, M. L.; DOS

SANTOS, D. C. 2009. Comparative morphology of the seminal receptacles of

Ocypode quadrata (Fabricius, 1787)(Brachyura, Ocypodoidea). Zootaxa, 2106: 41.

LÓPEZ GRECO, L. S.; LOPEZ, G. C.; RODRIGUEZ, E.M. 1999. Morphology of

spermatheca in the estuarine crab Chasmagnathus granulata Dana, 1851

(Graspidae, Sesarminae). Journal of Zoology, 249:490-493.

MCLAY, C. L. 1993. Crustacea Decapoda: the sponge crabs (Dromiidae) of New

Caledonia and the Philippines with a review of the genera. Mémoires du Muséum

national d'Histoire naturelle, 156:111-251.

MCLAY, C. L.; BECKER, C. 2015. Reproduction in brachyura. Treatise on zoology-

Anatomy, taxonomy, biology. The Crustacea, 9(Part C), 185-243.

MCLAY, C. L.; LOPEZ GRECO, L. S. 2011. A hypothesis about the origin of sperm

storage in the Eubrachyura, the effects of seminal receptacle structure on mating

strategies and the evolution of crab diversity: How did a race to be first become a

race to be last?. Zoologischer Anzeiger-A Journal of Comparative Zoology, 250:

378-406.

MELO, G. A. S. 1996. Manual de identificação dos Brachyura (caranguejos e Siris) do

litoral brasileiro. Editora Plêiade; Fundação de Amparo à Pesquisa do Estado de

São Paulo.

MINAGAWA, M. 1993. Gonopods of the red frog crab Ranina ranina Linnaeus

(Decapoda: Raninidae). Crustacean Research, 22: 45-54.

MINAGAWA, M., CHIU, J. R., KUDO, M.; TAKASHIMA, F. 1994. Male reproductive

biology of the red frog crab, Ranina ranina, off Hachijojima, Izu Islands, Japan.

Marine Biology, 118:393-401.

NAGAO, J.; MUNEHARA, H. 2003. Annual cycle of testicular maturation in the helmet

crab Telmessus cheiragonus. Fisheries Science, 69: 1200-1208.

NASCIMENTO, F. A. D.; ZARA, F. J. 2013. Development of the male reproductive

system in Callinectes ornatus Ordway, 1863 (Brachyura: Portunidae). Nauplius,

21:161-177.

NG, P. K. L.; GUINOT, D.; DAVIE, P. J. F. 2008. Systema Brachyurorum: Part 1. An

annotated checklist of extant Brachyuran crabs of the world. The Raffles Bulletin of

Zoology, 17: 1-286.

NICOLAU, C. F.; NASCIMENTO, A. A.; MACHADO-SANTOS, C.; SALES, A.; OSHIRO,

L. M. Y. 2012. Gonads of males and females of the mangrove tree crab Aratus


23 Caunesp

pisonii (Grapsidae: Brachyura: Decapoda): a histological and histochemical view.

Acta Zoologica, 93:222-230.

ROTLLANT, G.; GONZÁLEZ-GURRIARÁN, E.; FERNÁNDEZ, L.; BENHALIMA, K.;

RIBES. E. 2007. Ovarian maturation of the multi-spawning spider crab Maja

brachydactyla (Decapoda: Majidae) with special reference to yolk formation. Marine

Biology, 152: 383-394.

SANT'ANNA, B. S.; PINHEIRO, M. A. A.; MATAQUEIRO, M.; ZARA, F. J. 2007.

Spermathecae of the mangrove crab Ucides cordatus: a histological and

histochemical view. Journal of the Marine Biological Association of the United

Kingdom, 87: 903-911.

SIMEÓ, C. G.; RIBES, E; ROTLLANT, G. 2009. Internal anatomy and ultrastructure of

the male reproductive system of the spider crab Maja brachydactyla (Decapoda:

Brachyura). Tissue and Cell, 41:345-361.

SUBRAMONIAM, T. 1993. Spermatophores and sperm transfer in marine crustaceans.

Advances in marine biology, 29:129-214.

TAVARES, M.; SECRETAN, S. 1993. La notion de thelycum et de spermathèque chez les

Crustacés Décapodes. Comptes Rendus De l'Academie Des Sciences. Serie III, 316,

133-138.

TISEO, G. R.; MANTELATTO, F. L.; ZARA, F. J. 2014. Is cleistospermy and

coenospermy related to sperm transfer? A comparative study of the male

reproductive system of Pachygrapsus transversus and Pachygrapsus gracilis

(Brachyura: Grapsidae). Journal of Crustacean Biology, 34:704-716.

TISEO, G. R.; MANTELATTO, F. L.; ZARA, F. J. 2017. Ultrastructure of spermatophores

and spermatozoa of intertidal crabs Pachygrapsus transversus, Pachygrapsus

gracilis and Geograpsus lividus (Decapoda: Grapsidae). Zoologischer Anzeiger-A

Journal of Comparative Zoology, 269, 166-176.

TUDGE, C. Spermatozoal morphology and its bearing on decapod phylogeny. In: Martin,

J. W., Crandall, K.A.; Felder, D. L. 2009. (eds), Crustacean Issues 18: Decapod

Crustacean Phylogenetics. Boca Raton, Florida: Taylor & Francis/CRC Press. 101-

119.

ZARA, F. J.; PEREIRA, G. R. R.; SANT'ANNA, B. S. 2014. Morphological changes in the

seminal receptacle during ovarian development in the speckled swimming crab

Arenaeus cribrarius. The Biological Bulletin, 227: 19-32.

ZARA, F. J.; TOYAMA, M. H.; CAETANO, F. H.; LÓPEZ GRECO, L. S. 2012.

Spermatogenesis, Spermatophore, and Seminal Fluid Production in the adult Blue

Crab, Callinectes danae (Portunidae). Journal of Crustacean Biology, 32:249-262.

https://www.ncbi.nlm.nih.gov/labs/journals/c-r-acad-sci-iii/


Capítulo redigido de acordo com as normas do periódico Acta Zoologica Caunesp

Capítulo I

Sperm ultrastructure of four crab species of the family

Dromiidae (Crustacea: Brachyura): addition data to

Hypoconchinae and Dromiinae subfamilies

Maria Alice Garcia Bento, Ivana Trettin, Fernando L. Mantelatto & Fernando

José Zara


25 Caunesp

Sperm ultrastructure of four crab species of the family Dromiidae (Crustacea: Brachyura):

addition data to Hypoconchinae and Dromiinae subfamilies

Maria Alice Garcia Bento¹, Ivana Trettin¹, Fernando L. Mantelatto² & Fernando José Zara¹

¹ Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), FCAV, Departamento de

Biologia Aplicada, Laboratório de Morfologia de Invertebrados (IML), Via de Acesso Prof. Paulo

Donato Castellane, s/n, Jaboticabal, 14884-900, São Paulo, Brazil. E-mails:

[email protected]; [email protected].

²Laboratório de Bioecologia e Sistemática de Crustáceos, Departamento de Biologia, Faculdade de

Filosofia, Ciencias e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo (USP), Av.

Bandeirantes, 3900, CEP 14040-901, Ribeirão Preto, São Paulo, Brazil. E-mail: [email protected]

Condensed title: Ultrastructure of Dromiidae spermatozoa

mailto:[email protected]




26 Caunesp

ABSTRACT

We described the spermatozoa ultrastructure of Dromiidae Hypoconcha parasitica, H. arcuata,

Moreiradromia antillensis and Dromia erythropus and compared the morphologies among species

of Dromiidae, Dromioidea and Podotremata, to elucidate the relationship between different species

of this brachyuran group. Specimens were collected from the Northern coast of São Paulo, Brazil

and were fixed and processed following transmission electron microscopy routine. The Dromiidae

spermatozoa studied are characterized by discoid acrosome, with three or four concentric zones,

which are centrally separated by bilateral capitate perforatorial chamber, with the apex similar to

the "mushroom" shape in the Hypoconchinae and letter “T” in Dromiinae. Above of of the

perforatorial chamber there is an apical protuberance, continuous with the subopercular region and

the operculum, which forms a low dome, centrally perforated. Under differential interference

contrast phase, the spermatozoa show 3 to 4 radial arms. The spermatozoa characters in

Hypoconchinae and Dromiinae were not consistent to separate these subfamilies from the

Dromiidae and Dromioidea. The ultrastructural differentiation was only found between

representatives Dromiidae and Raninidae. Thus, the spermiotaxonomy corroborated the previous

morphological and molecular studies, supporting the monophyly of Dromiidae and Dynomenidae

in relation to Homolidae and Latreilliidae.

Correspondence: Maria Alice Garcia Bento, UNESP, Faculdade de Ciências Agrárias e

Veterinárias- Campus de Jaboticabal, Departamento de Biologia Aplicada, Laboratório de

Morfologia de Invertebrados (IML), Via de Acesso Prof. Paulo Donato Castellane, s/n,

Jaboticabal, 14884-900, São Paulo- SP, Brazil. E-mail: [email protected].


27 Caunesp

INTRODUCTION

Members of the family Dromiidae De Haan, 1833 are recognized by having intriguing

morphological differences when compared with the basic true crab design, which is considered as

primitive group within the species infraorder Brachyura, with approximately 50 recognized genera

distributed worldwide (Melo, 1996; Ng et al., 2008). These species are commonly known as

sponge crab or shellback crab by their association with shells of bivalves, sponges or ascidians

(Mclay, 1993; Melo, 1996; Melo and Campos, 1999; Guinot et al., 2013). This family comprises

the subfamilies Dromiinae, Hypoconchinae and Sphaerodromiinae, which are diagnosticated by

the peculiar organization of the thoracic sternal, abdomen, coxa of the fifth pereopod and penis in

males, and by the structure of the thoracic sternal 7/8 in females (Guinot and Tavares, 2003; Guinot

et al., 2013). In Brazil only representatives of the Hypoconchinae and Dromiinae are found,

represented by the two species of each subfamilies, Hypoconcha parasitica (Linnaeus, 1763), H.

arcuata Stimpson, 1858, Dromia erythropus (George Edwards, 1771), D. gouveai Melo and

Campos, 1999 and Moreiradromia antillensis (Stimpson, 1858) (Melo, 1996; Ng et al., 2008).

Regarding phylogeny, molecular works performed by Spears and Abele (1988) and Spears

et al. (1992) included the family Dromiidae as part of Anomura. Further, evidences indicated that

the larval morphology and the patterns of reproductive openings are more similar to Anomura

species. However, subsequent morphological revisions by McLay (1993), Guinot and Tavares

(2001; 2003), Guinot and Quenette (2005) and Guinot et al. (2013) and additional molecular

studies (Martin & Davis, 2001; Ahyong et al., 2007; Tsang et al., 2014) have refuted the

hypothesis of Dromiidae part of Anomura.

The study of the ultrastructure of spermatozoa has become an excellent tool in resolving

taxonomic issues that include relationship among species in several groups of decapod crustaceans

(Jamieson, 1994; Jamieson et al., 1995; Tudge, 1991; Tudge, 2009; Tudge et al., 2014).

Additionally, studies based on the comparison of the ultrastructure of the spermatozoa of different


28 Caunesp

species, allied with molecular phylogeny studies has given support to new proposals of phylogeny

to the group (Camargo et al., 2015, 2017; Assugeni et al., 2017). In Dromiidae, comparative

studies with spermatozoa structures under transmission electron microscopy were carried out only

with the species Dromidiopsis edwardsi Rathbun, 1919 and Stimdromia lateraris (=Petalomera)

Gray, 1831 from the subfamily Dromiinae and Sphaerodromia lamellata Crosnier, 1994 from the

subfamily Sphaerodromiinae (Jamieson et al., 1993a; Jamieson, 1994; Guinot et al., 1998).

However, there are no studies with representatives of Hypoconchinae subfamily and other

Dromiidae.

Therefore, considering the high diversity and the importance of this family as primitive

group and the scanty knowledge on spermiotaxonomy, we described the sperm ultrastructure of the

Hypoconchinae Hypoconcha parasitica, H. arcuata and of the Dromiinae Moreiradromia

antillensis and Dromia erythropus and compared their morphology with Dromiidae, Dromioidea

and Podotremata using literature data, in order to clarify the phylogenetic reproductive relationship

between the different species of Podotremata.

MATERIALS AND METHODS

Animal samples

Mature males of Hypoconcha parasitica, Hypoconcha arcuata, Moreiradromia antillensis

and Dromia erythropus were collected in the municipality of Ubatuba, São Paulo, Brazil

(25°07´385´´S/47°52´508´´W), from September 2014 to August 2016, using a double-rig shrimp

trawling. Subsequently, the animals were transported alive in Styrofoam boxes to the Invertebrate

Morphology Laboratory (IML) at the Department of Applied Biology for Agriculture, of the

Faculty of Agricultural and Veterinary Sciences at the Campus Jaboticabal at the São Paulo State

University (UNESP). The animals were identified according to Melo (1996).

Sperm ultrastructure


29 Caunesp

The animals were anesthetized by thermal shock (−20 oC) for 15 minutes (López-Greco et

al., 1999) and dissected. We used at least five animals per species, except D. erythropus of which

we collected only one specimen. Fragments of the posterior vas deferens (1mm³) were fixed in

Karnovsky fixative solution (2.5 % glutaraldehyde and 2 % paraformaldehyde) in a 0.1 M sodium

cacodylate buffer (pH 7.4), containing 5% sucrose for 4 hours (Ro et al., 1990). After fixation, the

samples were washed three times with the same buffer and post-fixed in 1% cacodylate-buffered

osmium tetroxide for 2 hours. The samples were “en bloc” stained with 1 % aqueous uranyl acetate

(overnight), dehydrated in an ascending acetone series (50 to 95 %), embedded and included in

Epon-Araldite. Thin and ultrathin section (50-60nm) were obtained with a Leica UC7

ultramicrotome, and contrasted with 2% uranyl acetate in water for 45 minutes and 0.4% lead

citrate in 0.1 N NaOH for 10 minutes. The samples were analyzed in JEOL-JEM 1010 transmission

electron microscope operated with an 80 kV electron beam, and the digital images were obtained

with Gatan camera in the Laboratory of Electron Microscopy, FCAV, UNESP – Jaboticabal

Campus, São Paulo, Brazil. For the measures of acrosome lenght/width (C:L), the spermatozoa

were arranged horizontally and the largest length and width of the acrosomal vesicle was used for

the proportion.

For analyses of the spermatozoa under differential interference contrast phase microscope

(DIC), the vas deferens fragments (1mm³) were maintained in sea water (pH 7.4) for the

preparation of temporary slides. For this, portions of the vas deferens were macerated on slides

containing ± 0.5 ml of sea water and covered with coverslips. These slides were observed in DIC

Zeiss Axio Imager Z2.

RESULTS

General diagram

The studied Dromiidae spermatozoa are typically podotreme in overall shape ultrastructure

(Fig. 1A). A large portion of the spermatozoon consists of the anteroposteriorly depressed


30 Caunesp

acrosome (Fig. 1A). The acrosome is discoid and the perforatorial chamber bilaterally capitate (Fig.

1A). Usually, four concentric horizontal zones are discernible occurring from the of the

perforatorial chamber in the acrosome vesicle, each one with their own peculiar features. The zones

are inner acrosome zone, ray acrosome zone, anterolateral electron-lucid zone and outer acrosome

zone (Fig. 1A). The anterior surface of the acrosome is domed over the opercuwlum (Fig. 1A). This

structure is perforated and centrally to this there is an apical protuberance with subopercular

material (Fig. 1A). The nucleus is electron-pale, filled with fibrous chromatin and, under DIC, are

observed two or three radial arms and the operculum perforation (Fig; 1A-D).

Species spermatozoa ultrastructure

Hypoconcha parasitica ─ In the vas deferens lumen, the spermatozoa are immersed in

moderately electron-dense secretion, surrounded by granular electron-dense secretion, without

typical spermatophore wall (Fig. 2A). Longitudinal section of entire spermatozoon in the wide

axis of the perforatorial chamber shows a bilateral capitate perforatorial chamber in the inner

acrosome, which has a slightly rounded apex resembling the form of a mushroom (Fig. 2B). The

nucleus is filled with fibrous chromatin, surrounding almost all the whole extent of the

acrosomal discoid vesicle (Fig. 2B). In longitudinal section the perforatorial chamber apex is

rounded (Fig. 2C) and, above the bilateral capitate perforatorial chamber, there is an apical

protuberance composed of subopercular material (Fig. 2D). The operculum is perforated

centrally and is discontinuous with the acrosomal capsule (Fig. 2D). The acrosome has the form

of a disc with C:L of 0.35 ± 0.05, and the horizontal zonation from the center to the margin

being divided into: (1) electron-dense innermost zone, surrounded the stalk of the bilateral

capitate perforatorial chamber; (2) outer acrosome zone, showing electron-dense granular

lamellae, surrounded near the apical apex of the perforatorial chamber and extending laterally

almost to the periphery of the acrosome; (3) acrosome ray zone, seated above the last zone and

(4) the anterolateral electron-lucid zone, that is an extension of the flange of the inner zone and

lies posteriorly to the acrosome ray zone (Fig. 2E). In the electron-lucid zone there is a sphere,


31 Caunesp

which is a discontinuous flange extension of the inner zone (Fig. 2F). The thin cytoplasm is

vestigial with reduced lamellar complex and mitochondria with little crest (Fig. 2G). The

nucleus is delimited by the nuclear envelope discontinuous with the acrosomal capsule (Fig.

2G). The cross section of the spermatozoon, near the anterior region of the perforatorial

chamber, confirms the bilaterally capitate morphology of this structure as well as the presence

of an inner acrosome zone, an acrosome ray zone and an anterolateral electron-lucid zone (Fig.

2H). The outer acrosome zone surrounds all the capitate edge of the perforatorial chamber and

the ray zone, in which it forms a circumference below the operculum (Fig. 2H). The base of the

perforatorial chamber is irregular, presenting three-pointed star-shape in a cross section near this

region (Fig. 2I). This cross section also exhibits the extensions of the inner layer of the

acrosome, the anterolateral electron-lucid zone and the nucleus (Fig. 2I).

Hypoconcha arcuata ─ the spermatozoa are immersed in moderately electron-dense type I

secretion, surrounded by more electron-dense secretion, without spermatophore wall (Fig. 3A).

The longitudinal section of entire spermatozoon in the wide axis of the perforatorial chamber

shows bilaterally capitate perforatorial chamber with rounded apex, resembling the form of a

mushroom with lamellae on the extremities (Fig. 3B). The perforatorial chamber showed

lamellae at the ends and the nucleus is filled with fibrous chromatin, which involves almost all

extension of the acrosomal vesicle (Fig. 3B). In longitudinal section in the narrow axis of the

perforatorial chamber, rounded apex is observed (Fig. 3C). Furthermore, cytoplasmic membrane

and mitochondria are present (Fig. 3C). Above the apical region of the perforatorial chamber

there is an apical protuberance, which is continuous with the subopercular region and the

operculum electron-dense centrally perforated (Fig. 3D). The acrosome is in shape of a disc,

with C:L of 0.3 ± 0.05, with horizontal zonation from the center to the periphery of the

acrosome vesicle, where four zones may be recognized. These are the innermost zone, which is

homogeneous, electron-dense and filled the lower region of the acrosome; the outer zone that is

less electron-dense adjacent to the perforatorial chamber; ray acrosome zone that is embedded

on the outer zone, and the anterolateral zone that is electron-lucid (Fig. 3E). The almost cross


32 Caunesp

section over the perforatorial chamber apex confirms the bilateral slightly rounded bilateral-

shaped of its extremities and shows the medial region between the operculum, the perforatorial

chamber, the outer acrosome zone and the ray acrosome zone (Fig. 3F).

Moreiradromia antillensis ─ The vas deferens lumen showed the spermatozoa immersed in

electron-dense secretion I, which is surrounded by electron-lucid secretion II with the presence

of electron-dense granules, without typical spermatophore wall (Fig. 4A). The longitudinal

section of the spermatozoon in the wide axis of the perforatorial chamber shows that it is

bilaterally capitate, with flattened apex, in the form of the letter “T” (Fig. 4B). The acrosome is

discoid and the nucleus is filled with fibrous chromatin (Fig. 4B). The longitudinal cross section

of the spermatozoon in the narrow axis of the axis confirms that it has a flat apex (Fig. 4C). The

operculum is centrally perforated and discontinuous with the acrosomal capsule (Fig. 4C - D).

The perforatorial chamber apex contains horizontal lamellae and above this there are the

operculum perforated and an apical protuberance filled with subopercular material (Fig. 4D).

The acrosome has the form of a disc, with C:L of 0.4 ± 0.05, showing horizontal zonation from

the center to the periphery, presenting an inner acrosome zone, composed by horizontal

striation; a homogeneous outer acrosome zone; an anterolateral electron-lucid zone, which

contains small portions of the inner zone (Fig. 4E). In cross section, it is possible to notice the

narrow stalk of the perforatorial chamber, the inner acrosome zone, the anterolateral electron-

lucid zone with small portions of inner zone and the nucleus (Fig. 4F).

Dromia erythropus ─ The spermatozoa are immersed in homogeneous, electron-dense type I

secretion (Fig. 5A). The spermatozoa and type I secretion are surrounded by type II secretion,

that it is moderately granular and electron-dense (Fig. 5A). The type II secretion is surrounded

by type III secretion, which is electron-dense (Fig. 5A). This latter secretion type is surrounded

by the type IV secretion that is heterogeneous and composed by two elements (Fig. 5A). One

element more abundant on the face in contact with type III secretion, which is less electron-

dense (Fig. 5A). The other element is next to the epithelium and is more electron-dense (Fig.

5A). The nucleus is filled with thin fibrous chromatin and the radial arms are not observed


33 Caunesp

under transmission electron microscopy (Fig. 5A-B). The acrosome has the form of a disc, with

C:L of 0.38 ± 0.02 (Fig. 5B-C). The longitudinal section above the wide axis of the perforatorial

chamber showed flat apex and, at the same time, confirms the bilaterally capitate morphology

forming a “T” shaped perforatorial chamber apex (Fig. 5B). The nucleus is filled with fibrous

chromatin (Fig. 5B). In the longitudinal section in the narrow axis, the perforatorial chamber is

thinly bilaterally capitate and rounded in its edges (Fig. 5C). The operculum form a dome-

shaped, with the thicker periphery and thinner centrally perforated inner edge and also the

discoid acrosome and mitochondria. (Fig. 5C). The perforatorial chamber presents lamellae,

which are perforatorium tubules (Fig. 5D). The operculum appears dome-shaped in longitudinal

section, with thicker external periphery and thinner centrally perforated inner margin (Fig. 5C-

D). The perforated subopercular region has an apical protuberance, which is subopercular

material spillage from the opercular perforation (Fig. 5D). The acrosome shows horizontal

zonation from the center to the periphery that is divided in four zones: the inner acrosome zone

that is moderately striated and adjacent to the base of the perforatorial chamber, the outer

homogeneous, electron-dense acrosome zone, located near the apical acrosome region, the ray

acrosome zone and the electron-lucid acrosome zone, both being close to the periphery (Fig.

5E). The anterolateral electron-lucid zone can be clearly observed at the edge of the acrosome in

longitudinal and transverse sections (Fig. 5E-F). The bilaterally capitate morphology becomes

clear in cross sections near the apex that forms a tetrahedral structure of the head of the

perforatorial chamber (Fig. 5F). We also observed that the anterolateral electron-lucid zone and

the acrosome ray zone surround the acrosome completely (Fig. 5F).

Dromiidae spermatozoa have discoid acrosome, with billaterally capitate perforatorial

chamber, perforated operculum, subopercular protuberance and two or three radial arms, as

observed in other species of Dromiidae described in the literature (Table 1). A comparison of

the fine ultrastructural characters of the spermatozoon among the Dromiidae species here

described and from the previous literature is provided in table 1.


34 Caunesp

DISCUSSION

Overall, the spermatozoa of Hypoconcha parasitica, H. arcuata, Moreiradromia antillensis

and Dromia erythropus followed the patterns found for both Dromiidae and Dromioidea species,

with the following synapomorphies: depression of the acrosome making it discoid, acrosome

zonation predominantly horizontal and perforated operculum. The capitate bilateral perforatorial

chamber is consider the unique apomorphy of Dromioidea (Jamieson, 1994).

The ultrastructural characters found in the spermatozoa of Hypoconchinae H. parasitica

and H. arcuata are more similar to each other than when compared to the Dromiinae species M.

antillensis and D. erythropus. The only differences found among the Hypoconchinae were in

relation to the outer acrosomal zone and to the presence of lamellae in the apex of the perforatorial

chamber. The outer acrosome zone of H. parasitica has lamellar aspect, while in H. arcuata, this

zone is homogeneous. In addition, the lamellae are present only at the apex of the perforatorial

chamber of H. arcuata. Also, the studied species of Dromiinae share more sperm characteristics

with species of this last subfamily, differing in the thickness of the operculum, presence of ray

zone, flange and capsular projections.

The spermatozoa of Hypoconchinae, when compared to other dromiids, showed more

similarity with the unique member described for the subfamily Sphaerodromiinae, Sphaerodromia

lamellata Crosnier, 1994. The only differences of H. parasitica were the operculum thickness,

presence of flange from the discontinuous inner acrosome zone on the anterolateral electron-lucid

zone and the wide outer zone that have lamellae resembling fingerprints, below the ray acrosome

zone, which does not occur in S. lamellata (Guinot et al., 1998). Moreover, the morphology of the

perforatorial chamber of the mushroom type not is identical, once in the median portion of H.

parasitica shows a narrowing, which does not occur in Sphaerodromiinae. When comparing

spermatozoa characters between H. arcuata and the Sphaerodromiinae subfamily, we noticed

differences in the operculum thickness and flange presence on the anterolateral electron-lucid zone.


35 Caunesp

The comparison of the spermatic characters among the Dromiinae shows that Dromiinae

present greater similarity with Dromidiopsis edwardsi (Jamieson et al., 1993a). The only

differences between M. antillensis and D. edwardsi were the C:L, the inner acrosome zone

constitution, the flange in the anterolateral electron-lucid zone, the radial arms and the capsular

projections. On the other hand, the differences between Dromia erythropus and Dromidiopsis.

edwardsi (Jamieson et al., 1993a) were only in relation to the thickness of the operculum, the

acrosome ray zone presence and in the constitution of the internal acrosomal zone.

The comparison between the spermatozoa of the Hypoconchinae and Dromiinae species

studied and the other species of Dromiinae, revealed that the main difference was related to the

acrosome ray zone, which is circular in species of Hypoconchinae. In M. antillensis and

Stimdromia lateralis (Gray, 1831) the acrosome ray zone is absent, while in Dromidiopsis

edwardsi an inconsistency in the descriptions were observed (Jamieson, 1994). Jamieson et al.

(1993c) described the acrosome ray zone, which is elongated in Dromidiopsis edwardsi, being this

zone later named intermediate zone (Jamieson, 1994). The varying thickness of the operculum was

found only in D. erythropus. Due to the morphological similarity in spermatic characters, our

results are in agreement with morphological based analysis of Hypoconchinae and

Sphaerodromiinae that suggest that these subfamilies are the most basal within Dromiidae

(Jamieson, 1994; Guinot & Tavares, 2003; Guinot et al., 2013).

When the Hypoconchinae species were compared with other representatives of

Dromioidea, we noted a great similarity of the spermatozoa ultrastructure between Dynomenidae

and Homolodromiidae (Jamieson et al., 1993a; Jamieson and Tudge, 2000). The only differences

between H. parasitica and Metadynomene tanensis (Yokoya, 1933) were the absence of

anterolateral electron-lucid zone, whereas Paradynomene tuberculata Sakai, 1963 compared with

H. parasitica shows the thinner inner zone (Jamieson et al., 1993a). The differences found between

H. arcuata and Met. tanensis are regarding C:L, thickness of the operculum, presence of the flange,

lamellae at the apex of the perforatorial chamber and radial arms, whereas the differences between


36 Caunesp

this Hypoconchinae and P. tuberculata are thickness of the operculum and presence of lamellae at

the apex of the of the perforatorial chamber (Jamieson et al., 1993a).

The comparison among the studied Dromiinae and the Dromioidea from the literature

shows that D. erythropus shares more sperm characters with the species of Dynomenidae than with

those of Homolodromiidae. The differences found between D. erythropus and Met. tanensis were

the acrosome C:L ratio, the constitution of the internal acrosomal zone, the perforatorial chamber

apex and the bilaterally capitate perforatorium chamber figure. While D. erythropus and P.

tuberculata differ in the constitution of the internal acrosomal zone, presence of the flange, the

perforatorial chamber apex and bilaterally capitate perforatorium chamber. The spermatozoon of

M. antillensis is more distinct from species of Dynomenidae and Homolodromiidae and all

characteristics of M. antillensis were observed in the spermatozoa of other Dromiidae species from

the literature (Jamieson et al., 1993 a, b, c; Jamieson, 1994, Guinot et al., 1998, Jamieson and

Tudge, 2000).

According to Jamieson and Tudge (2000), the spermatic characteristics found in

Homolodromiidae represent a mix between the characters of Dromiidae and Dynomenidae. We

also observed this mix of characters and therefore could not yet find any robust character to

separate the Dromiidae from this study from species of Homolodromiidae. Thus, there is no distinct

pattern of the ultrastructure of the spermatozoa for Dromioidea, and the representatives of this

superfamily only differ from others Podotremata, which agrees with the proposal from Guinot et al.

(1998). Moreover, the absence of distinct characters between Dromioidea families and subfamilies

is in agreement with the monophyly of the group, previously proposed by means of

spermiotaxonomy (Jamieson, 1994, Jamieson et al., 1995, Guinot et al., 1998) and molecular

analyzes (Ahyong et al., 2007; Tsang et al., 2014). Although Ahyong et al. (2007) suggests that

Hypoconcha is the most divergent branch within the topology for Dromiidae, no spermatic

characteristic was observed to corroborate this finding. Furthermore, in this last studied the only

representative of Dromiinae is closer to Dynomenidae in the phylogenetic tree. Regardless, the

studied species of Hypoconchinae and Dromiinae have typically dromioid spermatozoa and any


37 Caunesp

attempt to include them in Anomura (Spears et al., 1992), must be abandoned and has no

spermiotaxonomy support.

Tsang et al. (2014) demonstrated that Dromiidae and Dynomenidae are a monophyletic

group, as well as Homolidae and Latreilliidae are also monophyletic. Additionally, due to the

addition of Latreilliidae representatives these two groups are shown to be paraphyletic (Tsang et

al., 2014). When we compared H. parasitica, H. arcuata, M. antillensis and D. erythropus as well

as the Dromiidae and Dynomenidae described in the literature, we noticed a clear separation of

sperm characteristics between the two clades. Homolidae and Latreilliidae, differ in

spermiotaxonomy from each other and between Dromiidae and Dynomenidae, in the following

characters: 1) the acrosome with less discoid-shape, 2) the apical protuberance undeveloped

(Homolidae) or absent (Latreilliidae), 3) the capitate perforatorium chamber, 4) absence of the

acrosome ray zone and 5) opercular projections (Homolidae). Although molecular data for

Homolodromiidae were not included in the most current phylogeny of Brachyura (Tsang et al.,

2014), based on the ultrastructure of spermatozoa, it is clear to us that Homolodromiidae is more

related to Dromiidae and Dynomenidae than Homolidae and Latreilliidae.

When the spermatozoa of the studied Dromiidae was compared to the Podotremata from

the Raninidae family, it was noticed a pronounced difference of structural organization (Jamieson,

1989; Jamieson and Tudge, 2000). In Raninidae, the spermatozoa shows another type of acrosomal

zonation, which is more concentric than horizontal, presence of multiple capsular projections,

posterior capsule chambers, tapered and reduced perforatorium chamber and specially, posterior

median process (Jamieson, 1989), typical for Majoidea (for revision see Tudge et al., 2012). In a

general analysis, the spermatozoon of R. ranina shared more characteristics with representatives of

Cyclodorippoidea, Homolidae and Eubrachyura than with Dromiidae or other Dromioidea, which

cohoborates what is observed in molecular phylogeny (Jamieson and Tudge, 2000; Tsang et al.,

2014). Thus, the proposed grouping using of Dromiacea, Raninoidea and Cyclodorippoidea,

supported by molecular phylogeny (Ahyong et al., 2007, 2011), appear to better represent the

morphological variations of the spermatozoa ultrastructure than the use of Podotremata. Therefore,


38 Caunesp

the spermatozoa ultrastructural characteristics are a robust tool that could be essential for the

resolution of taxonomic problems, in association with molecular tools.

ACKNOWLEDGMENTS

The present study was supported by a Master Fellowship from the Research São Paulo

Foundation FAPESP (grant # 2016/10393-4 to MAGB), as part of the multidisciplinary research

projects BIOTA – Research São Paulo Foundation FAPESP (grant # 2010/50188-8) and the

Coordination for the Improvement of Higher Education Personnel (CAPES) - Programa

Ciências do Mar II – CIMAR (#1989/2014-23038.004309/2014-51, #2005/2014-

23038.004308/2014-14) granted to FLM and FJZ. FJZ and FLM are thankful to the Conselho

Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Universal #486337/2013-8

to FJZ and PQ 304968/2014-5 to FLM). The authors would also like to thank the Electron

Microscopy Laboratory of FCAV UNESP – Jaboticabal facility. This study was conducted in

accordance with Brazilian laws (FJZ-MMA SisBio permanent license No. 34587-1; permanent

license to FLM for the collection of Zoological Material No. 11777-1 MMA/IBAMA/SISBIO).

REFERENCES

AHYOUNG, T. S., LAI, J. C. Y., SHARKEY, D., COLGAN, D. J., & NG, P. K. L. (2007).

Phylogenetics of the brachyuran crabs (Crustacea: Decapoda): The status of Podotremata

based on small subunit nuclear ribosomal RNA. Molecular Phylogenetics and Evolution, 45,

576-586.

AHYONG, S. T., LOWRY, J. K., ALONSO, M., BAMBER, R. N., BOXSHALL, G. A.,

CASTRO, P., GERKEN, S., KARAMAN, G. S., GOY, J. W., JONES, D. S., MELANDA,

K., ROGERS, D. C., & SVAVARSSON, J. (2011). Subphylum Crustacea Brünnich, 1772.

In: Zhang Z-Q, editor. Animal biodiversity: an outline of higher-level classification and

survey of taxonomic richness. Zootaxa, 3148, 165-191.

BENETTI, A. S., SANTOS, D. C., NEGREIROS-FRANSOZO, M. L., & SCELZO, M. A. (2008).

Spermatozoal ultrastructure in three species of the genus Uca Leach, 1814 (Crustacea,

Brachyura, Ocypodidae). Micron, 39, 337-343.


39 Caunesp

CAMARGO, T. R., ROSSI, N., CASTILHO, A. L., COSTA, R. C., MANTELATTO, F. L., &

ZARA, F. J. (2016). Integrative analysis of sperm ultrastructure and molecular genetics

supports the phylogenetic positioning of the sympatric rock shrimps Sicyonia dorsalis and

Sicyonia typica (Decapoda, Sicyoniidae). Zoomorphology, 135, 67-81.

GUINOT, D., JAMIESON, B. G. M., & FORGES, B. R. (1998). Comparative spermatozoal

ultrastructure of the three Dromiacean families exemplified by Homolodromia kai

(Homolodromiidae), Sphaerodromia lamellata (Dromiidae) and Dynomene tanensis

(Dynomenidae) (Podotremata: Brachyura). Journal of Crustacean Biology, 18, 78-94.

GUINOT, D., & QUENETTE, G. (2005). The spermatheca in Podotreme crabs (Crustacea,

Decapoda, Brachyura, Podotremata) and its phylogenetic implication. Zoosystema, 27, 267-

342.

GUINOT, D., & TAVARES, M. (2001). Une nouvelle famille de crabes du Crétacé, et la notion de

Podotremata Guinot, 1977 (Crustacea, Decapoda, Brachyura). Zoosystema, 23, 507-546.

GUINOT, D., & TAVARES, M. (2003). A new subfamilial arrangement for the Dromiidae de

Haan, 1833, with diagnoses and descriptions of new genera and species. Zoosystema, 25, 43-

129.

GUINOT, D., TAVARES, M., & CASTRO, P. (2013). Significance of the sexual openings

and supplementary structures on the phylogeny of brachyuran crabs (Crustacea,

Decapoda, Brachyura), with new nomina for higher-ranked podotreme taxa. Zootaxa,

3665, 1- 414.

HINSCH, G. W. (1986). A comparison of sperm morphologies, transfer and sperm mass storage

between two species of crab, Ovalipes ocellatus and Libinia emarginata. International

Journal of Invertebrate Reproduction and Development, 10, 79-87.

JAMIESON, B. G. M. (1994). Phylogeny of the Brachyura with Particular Reference to the

Podotremata: Evidence from a Review of Spermatozoal Ultrastructure (Crustacea,

Decapoda). Philosophical Transactions: Biological Sciences, 345, 373-393.

JAMIESON, B. G. M., GUINOT, D., & FORGES, B. R. (1993b). The ultrastructure of the

spermatozoon of Paradynomene tuberculata Sakai, 1963 (Crustacea, Brachyura,

Dynomenidae): synapomorphies with dromiid sperm. Helgolander Meeresuntersuchungen,

47, 311-322.

JAMIESON, B. G. M., GUINOT, D., & FORGES, B. R. (1993c). Spermatozoal ultrastructure in

four genera of Homolidae (crustacea, decapoda): Exemplified by Homologenus sp.,


40 Caunesp

Latreillopsis sp., Homolomannia sibogae and Paromolosis boasi. Helgoländer

Meeresuntersuchungen, 47, 323-334.

JAMIESON, B. G. M., GUINOT, D., & FORGES, B. R. (1995). Phylogeny of the Brachyura

(Crustacea, Decapoda): evidence from spermatozoal ultrastructure. In: Jamieson, B. G. M.,

Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy.

Muséum National d’ Histoire Naturelle, 166, 265-283.

JAMIESON, B. G. M., & TUDGE, C. C. (2000). Crustacea-Decapoda. In: Jamieson,

B.G.M. (Ed.), Progress in Male Gamete Ultrastructure and Phylogeny, 1-95.

JAMIESON, B. G. M., TUDGE, C. C., & SCHELTINGA, D. M. (1993a). The ultrastructure of the

Spermatozoon of Dromidiopsis edwarsi Rathbun, 1919 (Crustacea: Brachyura: Dromiidae):

Confirmation of a Dromiid Sperm Type. Australian Journal of Zoology, 41, 537-48.

KLAUS, S., SCHUBART, C. D., & BRANDIS, D. (2009). Ultrastructure of Spermatozoa and

Spermatophores of Old World Freshwater Crabs (Brachyura: Potamoidea: Gecarcinucidae,

Potamidae, and Potamonautidae). Journal of Morphology, 270, 175-193.

MARTIN, J. W., & DAVIS, G. E. (2001). An updated classification of the recent Crustacea.

Natural History Museum of Los Angeles Country, 1-124.

MCLAY, C. L. (1993). Crustacea Decapoda: the sponge crabs (Dromiidae) of New Caledonia and

the Philippines with a review of the genera. Mémoires du Muséum national d'Histoire

naturelle, 156, 111-251.

MELO, G. A. S. (1996). Manual de identificação dos Brachyura (caranguejos e Siris) do litoral

brasileiro. Editora Plêiade; Fundação de Amparo à Pesquisa do Estado de São Paulo.

MELO, G. D., & CAMPOS JR, O. (1999). A família Dromiidae De Haan no litoral brasileiro, com

descrição de uma nova espécie (Crustacea: Decapoda: Brachyura). Revista Brasileira de

Zoologia, 16, 273-291.

NG, P. K. L., GUINOT, D., & DAVIE, P. J. F. (2008). Systema Brachyurorum: Part 1. An


Zoology, 17, 1-286.

REYNOLDS, E. S. (1963). The use of citrate at high pH as an eléctron opaque stain in eléctron

microscopy. The Journal of Cell Biology, 17, 208.


41 Caunesp

RO, S., TALBOT, P., TRUJILLO, J. L., & LAWRENCE, A. L. (1990). Structure and function of

the vas deferens in the shrimp Penaeus setiferus: segments 1-3. Journal of Crustacean

Biology, 10, 455-468.

SPEARS, T., & ABELE, L. G. (1988). Molecular phylogeny of brachyuran crustaceans based on

18S RNA nucleotide sequences. American Zoologist, 28, 2A.[Abstract].

SPEARS, T., ABELE, L. G., & KIM, W. 1992. (1992). The monophyly of brachyuran crabs: a

phylogenetic study based on 18S rRNA. Systematic Biology, 41, 446-461.

TSANG, L. M., SCHUBART, C. D., AHYONG, S. T., LAI, J. C., AU, E. Y., CHAN, T. Y., NG,

K. L. P., & CHU, K. H. (2014). Evolutionary history of true crabs (Crustacea: Decapoda:

Brachyura) and the origin of freshwater crabs. Molecular biology and evolution, 31, 1173-

1187.

TUDGE, C. C. (1991). Spermatophore Diversity Within and Among the Hermit Crab Families,

Coenobitidae, Diogenidae, and Paguridae (Paguroidea, Anomura, Decapoda). The Biological

Bulletin, 181, 238-247.

TUDGE, C. C. (2009). Spermatozoal morphology and its bearing on decapod phylogeny. In:

Martin, J.W., Crandall, K.A. & Felder, D.L. (eds), Crustacean Issues 18: Decapod

Crustacean Phylogenetics. Boca Raton, Florida: Taylor & Francis/CRC Press, 101-119.

TUDGE, C. C. , & KOENEMANN, S. (2009). Spermatozoal morphology and its bearing on

decapod phylogeny. In: Martin JW, Crandall KA, Felder DL, editors. Decapod Crustacean

Phylogenetics. Crustacean Issue 18. Baton Rouge, FL: CRC Press. 101-120.

TUDGE, C. C., SCHELTINGA, D. M., JAMIESON, B. G., GUINOT, D., & RICHER DE

FORGES, B. (2014). Comparative ultrastructure of the spermatozoa of the Majoidea

(Crustacea, Decapoda, Brachyura) with new data on six species in five genera. Acta

Zoologica, 95, 1-20.


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List of legends


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Figure 1. General diagram of Dromiidae spermatozoa under transmission electron

microscope and differential interference contrast microscopy (DIC). (A) The

spermatozoa have discoid acrosome, with bilaterally capitate perforatorial chamber. Note

four concentric horizontal zones occurring from the perforatorial chamber, such as: inner

acrosome zone, acrosome ray zone, anterolateral electron-lucid zone and outer acrosome

zone. The operculum showed perforation and centrally to this there is an apical

protuberance. The nuclei are filled with fibers of chromatin, with the presence or absence

of radial arms. (B) Spermatozoa of H. parasitica and H. arcuata on DIC showing the

operculum centrally perforated and the presence of two or three radial arms. (C) On DIC,

the spermatozoa of M. antillensis and (D) D. erythropus show an operculum and three

radial arms. RA = radial arms; O = operculum.

Figure 2. Spermatozoa ultrastructure of Hypoconcha parasitica. (A) Lumen of the

posterior vas deferens showing the spermatozoa immersed in secretion moderately

electron-dense, surrounded by the more electron-dense secretion, without the typical

spermatophore wall. (B) Longitudinal section of entire spermatozoon in the wide axis of

the perforatorial chamber, showing their bilateral capitate morphology and apex with a

mushroom-shape. Notice the thin nucleus filled with fibrous chromatin, surrounding

almost all the whole extent of the acrosomal discoid vesicle. (C) In longitudinal section,

the apex of the perforatorial chamber is rounded. Observe the acrosome and the nucleus

filled with fibrous chromatin. (D) Above the perforatorial chamber, there is the continuous

apical protuberance with the subopercular region and the operculum is perforated (arrow)

and discontinuous with the acrosomal capsule. (E) The acrosome zones are electron-dense

innermost zone, surrounded the stalk of the perforatorial chamber; outer acrosome zone,

showing electron-dense granular lamellae and surrounded near the apical apex of the

perforatorial chamber; acrosome ray zone, seated above the last zone and the anterolateral

electron-lucid zone, lies posterior to the acrosome ray zone. (F) The sphere on the

anterolateral electron-lucid zone (arrow) is an extension of the flange of the inner zone.

Note the acrosome ray zone and a little portion of the anterolateral electron-lucid zone. (G)

Detail of the thin cytoplasm near the edge of the acrosome, showing a mitochondria with


44 Caunesp

little crest and the nucleus delimited by the nuclear envelope (arrow) discontinuous with

the acrosomal capsule. (H) Cross section of the spermatozoon, near the anterior region of

the perforatorial chamber, showing the bilaterally capitate morphology of this structure

(arrows), inner acrosome zone, acrosome ray zone and anterolateral electron-lucid zone.

Note that the outer acrosome zone surrounds all the capitate edge of the perforatorial

chamber region and the ray zone, in which it forms a circumference below the operculum.

(I) The irregular base of the perforatorial chamber, showing three-pointed star-shape in the

cross section near this region. The base of the perfuratorium becames wider and forms an

irregular figure (arrow). This cross section also shows the extensions of the inner layer of

the acrosome, the anterolateral electron-lucid and the nucleus. AC = acrosome; AP = apical

protuberance; ARY = ray acrosome zone; EA = anterolateral electron-lucid zone; IA =

inner acrosome zone; M = mitochondria; N = nucleus; O = operculum; OA = outer

acrosome zone; P = perforatorial chamber; PA = perforatorial chamber apex; PB =

perforatorial chamber base; SI = type I secretion; SII = type II secretion; SZ =

spermatozoa.

Figure 3. Spermatozoa ultrastructure of Hypoconcha arcuata. (A) The spermatozoa are

immersed in less electron-dense type I secretion, which are surrounded by more electron-

dense type II secretion. Note that typical spermatophore wall is absent. (B) Overview of

spermatozoa in longitudinal section on the wide axis of the bilaterally capitate perforatorial

chamber, showing the mushroom-shaped morphology of the apex. Observe the presence of

lamellae at the ends of the perforatorial chamber and the nucleus filled with fibrous

chromatin, which involves almost all extension of the acrosomal vesicle. (C) In

longitudinal section of the narrow axis of the perforatorial chamber, their apex is rounded.

Note the cytoplasmic membrane and mitochondria. (D) Above the perforatorial chamber is

the apical protuberance and the operculum electron-dense centrally perforated. (E) The

discoid acrosome composed of four concentric zones, such as: homogeneous and electron-

dense inner acrosome zone, filled the lower region of the acrosome; the less electron-dense

outer acrosome zone, adjacent to the perforatorial chamber apex; acrosome ray zone,

embedded on the outer zone and the anterolateral electron-lucid zone. (F) The almost

transverse cross over the apex of the perforatorial chamber (white arrows) confirms the

slightly rounded bilateral-shape of its extremities and we can observe the anterolateral

electron-lucid region (black arrows) between the perforatorial chamber and the outer


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acrosome zone, and the ray acrosome zone. AC = acrossome; AP = apical protuberance;

ARY = ray acrosome zone; CY = cytoplasm; EA = anterolateral electron-lucid zone; IA =

inner acrosome zone; M = mitochondria; N = nucleus; L = lamellae; O = operculum; OA=

outer acrosome zone; P = perforatorial chamber; PA = perforatorial chamber apex; SI =

type I secretion; SII = type II secretion; SZ = spermatozoa.

Figure 4. Spermatozoa ultrastructure of Moreiradromia antillensis. (A) General

appearance of the vas deferens lumen, showing spermatozoa in electron-dense secretion I,

which is involved by the less electron-lucid secretion II with the presence of electron-dense

granules, without the typical spermatophore wall. (B) The longitudinal cross in the wide

axis of the perforatorial chamber shows their bilateral capitate morphology, with flattened

apex, shaped similarly to the letter “T”. Note the discoid acrosome and the nucleus filled

with fibrous chromatin. (C) Longitudinal section in the narrow axis of the perforatorial

chamber showing the flat apex and the centrally perforated operculum, which is

discontinuous with the acrosomal capsule (arrow). (D) Perforatorial chamber apex with

horizontal lamellae. Note the operculum (arrowhead) and the apical protuberance filled

with subopercular material. (E) The discoid acrosome showing the horizontal zonation is

formed by four zones: the inner acrosome zone, composed by horizontal striation (write

arrow), the homogeneous outer acrosome zone, the anterolateral electron-lucid zone, which

seems to contain electron-dense remnants of the inner acrosome zone (black arrow). (F) In

cross section, note the narrow stalk of the perforatorial chamber, the inner acrosome zone,

anterolateral electron-lucid zone with small portions of inner zone and the nucleus. AC =

acrosome; AP = apical protuberance; EA = anterolateral electron-lucid zone;; IA = inner

acrosome zone; N = nucleus; L = lamellae; O = operculum; OA = outer acrosome zone; P

= perforatorial chamber; PA = perforatorial chamber apex; SI = type I secretion; SII = type

II secretion; SZ = spermatozoa.

Figure 5. Spermatozoa ultrastructure of Dromia erythropus. (A) The spermatozoa are

immersed in electron-dense, homogeneous type I secretion, which is surrounded by the

type II secretion, that is moderately electron-dense with granules. The II secretion is

involved by electron-dense type III secretion. Note that this latter secretion is surrounded

by heterogeneous type IV secretion, composed of two elements: one more abundant on the


46 Caunesp

face in contact with type III secretion, which is less electron-dense and another, next to the

epithelium, which is less electron-dense (arrows). (B) Discoid acrosome in longitudinal

section to the wide axis of the bilaterally capitate perforatorial chamber, showing the

morphology with the shape of the letter "T", with flat apex. Note the nucleus filled with

fibrous chromatin. (C) Longitudinal section in the narrow axis of the perforatorial

chamber, showing that it is thinly bilaterally capitate and rounded in the edges. In this

section, also note the presence of the operculum forming a dome-shaped, with the thicker

periphery (white arrowhead) and thinner centrally perforated inner edge (black arrowhead),

and also the discoid acrosome and mitochondria. (D) Opercular region perforated

(arrowhead) marked with the apical protuberance and the perforatorial chamber apex

showing horizontal lamellae. (E) Acrosome with horizontal zonation, being divided in

inner acrosome zone slightly striated, adjacent to the base of the perforatorial chamber,

electron-dense, homogeneous outer acrosome zone, near the apical region of the acrosome,

ray acrosome zone and anterolateral electron-lucid zone. (F) Morphology of the bilaterally

capitate perforatorial chamber in cross section near the apex, forming a tetrahedral

structure. Note the anterolateral electron-lucid and ray acrosome zones surrounded

completely the acrosome. AC = acrosome; AP = apical protuberance; ARY = ray acrosome

zone; EA = anterolateral electron-lucid zone; IA = inner acrosome zone; M =

mitochondria; N = nucleus; L = lamellae; O = operculum; Ao = outer acrosome zone; P =

perforatorial chamber; PA = perforatorium chamber apex; SI = type I secretion; SII = Type

II secretion; SIII = type III secretion; SIV = type IV secretion; SZ = spermatozoa.


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List of figures and table


48 Caunesp

Fig. 1


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Fig. 2


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Fig. 3


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Fig. 4


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Fig. 5


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¹

Table 1. Comparison of the ultrastructural characters of the spermatozoid among species of the Dromiidae subfamilies, described in the literature and from this study.

Hypoconchinae Dromiinae Sphaerodromiinae

Characteristics Hypoconcha

parasitica¹

Hypoconcha

arcuata¹

Dromia

erythropus¹

Dromidiopsis

Edwardsi4,5

Moreiradromia

antillensis¹

Stimdromia

Lateralis2,3,5

Sphaerodromia

lamellata2,5

Length/ width 0.3 0.4 0.5

Acrosome

Anterolateral electron-lucid zone Present

Ray acrosome zone – rounded on the

outer acrosome zone

Present

Absent

Present

Outer acrosome zone Lamellar Homogeneous

Inner acrosome zone Homogeneous Striated Homogeneous Striated Homogeneous

Flange of the inner acrosomal layer

discontinuous in the electron-lucid

zone

Present

Absent

Present

Absent

Aspect of chromatin Granular

Capsular projections Absent Present Absent

Centriole Present Absent Present Absent

Lamellar structure Present

Mitochondria Present

Operculum

Type Perforate

Opercular projections Absent

Continuity of the capsule and

operculum

Discontinuous

Operculum thickness Thin Thick and thin Thin Thick

Subopercular protuberance Present

Perforatorial

chamber

Perforatorial chamber apex Rounded Flat Rounded

Bilateral capitate perforatorial

chamber (figure of)

Mushroom Letter “T” Mushroom

Lamellae at perforatorial chamber

apex

Absent Present Absent Present

Radial arms Present ¹This study, ²Jamieson (1994); ³Jamieson et al (1993); 4 Guinot et al (1998); 5Jamieson et al., (1995)


Capítulo redigido de acordo com as normas do periódico Zoological Journal of the Linnean Society

Caunesp

Capítulo II

SEMINAL FLUID PRODUCTION AND SPERM TRANSFER IN

DROMIIDS: NEW INSIGHTS INTO THE EVOLUTION OF CRAB

REPRODUCTION

Maria Alice Garcia Bento, Laura López Greco and Fernando José Zara


55 Caunesp

SEMINAL FLUID PRODUCTION AND SPERM TRANSFER IN DROMIIDS: NEW INSIGHTS INTO

THE EVOLUTION OF CRAB REPRODUCTION

Maria Alice Garcia Bento1, Laura López Greco

2 and Fernando José Zara

1

1 Univ. Estadual Paulista (UNESP), FCAV, Invertebrate Morphology Laboratory (IML),

Departamento de Biologia Aplicada à Agropecuária and Aquaculture Center (CAUNESP), Via de

Acesso Prof. Paulo Donato Castellane, s/n, Jaboticabal, 14884-900, São Paulo, Brazil;

2 Universidad de Buenos Aires. CONICET. Instituto de Biodiversidad y Biología Experimental y

Aplicada (IBBEA). Facultad de Ciencias Exactas y Naturales, Departamento de Biodiversidad y

Biología Experimental, Laboratorio de Biología de la Reproducción y el Crecimiento de Crustáceos

Decápodos, C1428EGA, Buenos Aires, Argentina.

*Corresponding author. E-mail: [email protected]

Short running title: SPERMATOPHORE PRODUCTION AND SPERM TRANSFER IN

DROMIIDAE CRABS



56 Caunesp

ABSTRACT

Reproductive anatomy, including sperm storage structures and sperm transfer, is an important feature

used to analyze phylogenetic relationships among taxa. We describe male reproductive anatomy, the

seminal fluid production and the new spermatozoa packaging in the vas deferens of primitive crabs,

also emphasizing spermatozoa transfer compared to true crabs. In all species of Dromiidae, the testes

were tubular type and the vas deferens is a tubule with a simple epithelium. The spermatozoa are in a

central mass immersed in type I secretion, forming a large spermatic cord. In Hypoconchinae species

and Moreiradromia antillensis, the spermatic cord is surrounded by type II secretions, while in D.

erythropus we also found secretions of types III and IV. These patterns produce a unique elongated

kind of coenospermic “spermatophore,” or continuous cord totally different from the spermatophores

of all true crabs. In all Dromiidae species, the penial tube is a long conical-round paired organ. The

apex of penial tubes has a mobile operculum, avoiding the reflux from gonopod G2 during the sperm

transfer. A true sperm plug was not found in all studied species. Our results show a different pattern to

sperm package and new insights about how the sperm transfer occurs in Podotremata.

ADDITIONAL KEYWORDS: anatomy – ultrastructure – male reproductive system – Hypoconcha –

Podotremata – spermatheca.


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INTRODUCTION

Detailed comparative morphological analyses of the reproductive systems and

associated structures of Decapoda allow the use of these characters for phylogenetic and

evolutionary inferences (McLay & López Greco, 2011; Guinot, Tavares & Castro, 2013;

López Greco, 2013; McLay & Becker, 2015). Primitive crabs are included in Podotremata

(Guinot, 1977). In the species in this group, the paired gonopores are sternal located on the

coxae of both fifth pereopods of males and on the coxae of each third pereopod in females

(Guinot & Tavares, 2003, Guinot & Quenette, 2005; Guinot et al., 2013). In addition, the

females exhibit paired spermathecae, which are a enlarged intersegmental phragmata

formed by adjacent boundaries of sternites 7 and 8, with the opening in different sternites,

depending on the family or subfamily (Tavares & Secretan, 1993; Guinot & Tavares, 2003;

Guinot & Quenette, 2005). The spermatheca is a structure of ectodermal origin in which

the spermatozoa are stored (Hartnoll, 1969; Guinot & Quenette, 2005; Guinot et al., 2013).

This structure has no connection to the ovaries; therefore, the fertilization process occurs

outside the body (Hartnoll, 1969; Guinot & Quenette, 2005; Guinot et al., 2013). In

contrast, in the true crabs (eubrachyuran), the gonopores are sternal, and the spermatozoa

are stored in the pair of seminal receptacles located within the sixth thoracic sternite, with

direct connection to the ovaries by the oviducts (for revision McLay & López Greco, 2011;

McLay & Becker, 2015). Thus, the fertilization in these latter crabs takes place inside the

seminal receptacles (Hartnoll, 1969; McLay & López Greco, 2011).

Generally, the male reproductive system of crabs is a bilateral organ forming an “H”

shape that is composed of a pair of testis continuous with a pair of vasa deferentia ending

in the posterior region of the pair of fifth pereopods (Krol, Hawkins & Overstreet, 1992).

Spermatogenesis and spermiogenesis occur in the testes, and from a histological point of

view, this organ can be classified as lobular or tubular (Nagao & Munehara, 2003). Mature

spermatozoa are released in the vas deferens, which can be divided into three regions based

on morphology and function: anterior (AVD), where the spermatophores are formed; a

middle region (MVD); and a posterior region (PVD), where seminal fluid production

occurs, surrounding the spermatozoa until copulation (Krol et al., 1992; Zara et al., 2012;

Tiseo, Mantelatto & Zara, 2014). Additionally, the MVD and PVD of some brachyuran

species present accessory glands, also called outpockets, diverticula or caeca (Johnson,

1980; Simeó, Ribes & Rotllant, 2009; Tiseo et al., 2014). These structures produce

different types of secretions, which are added to the seminal fluid (Simeó et al., 2009,

Tiseo et al., 2014).


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In Decapoda, spermatozoa are packaged in different ways inside the vas deferens. In

lobsters (Astacidea), there are different secretions sequentially produced along the vas

deferens constituting the spermatophore wall (for revision Kooda-Cisco & Talbot, 1982;

Dudenhausaennd & Talbot; 1983; Hobbs, Harvey & Hobbs, 2007; López Greco, Vazquez

& Rodríguez, 2007; López Greco, 2013). In hermit crabs (Anomura) the spermatozoa are

packed into complex spermatophores composed of an ampulla, peduncle, stalk and

pedestal (Hinsch, 1991; Tudge, 1991; Mantelatto, Scelzo & Tudge, 2009; Fantucci &

Mantelatto, 2011). In Brachyura, the spermatozoa are packaged into elliptical

coenospermic spermatophores, in which several spermatozoa are grouped and delimited by

a single wall (El-Sherief, 1991; Guinot, Jamieson & Tudge, 1997; Anilkumar, Sudha &

Subramoniam, 1999; Tiseo et al., 2014; Tiseo, Mantelatto & Zara, 2017). In contrast, in

some freshwater crabs and one marine species, each spermatophore has only one

spermatozoon, known as a cleistoespermic spermatophore (Guinot et al., 1997; Klaus,

Schubart & Brandis, 2009; Tiseo et al., 2014, 2017). The absence of spermatophores is a

rare condition in Brachyura, but it has been observed in some freshwater crabs from

Potamidae and Gecarcinucidae (Guinot et al., 1997; Klaus et al., 2009; Klaus & Brandis,

2011). In Podotremata, few articles have described the pattern of sperm packaging within

the vas deferens. In Dromiidae and Raninidae, the male reproductive system is divided into

three anatomical regions (Hartnoll, 1975; Minagawa et al., 1994), while in Homolidae, no

different regions are described, despite them being obvious in the drawings of Hartnoll

(1975). The histological differences schematized for Dromia personata (Linnaeus, 1758)

need to be reviewed in Dromiidae since the anterior and median regions seem to be very

similar in the drawings. In studies of Podotremata, the authors do not discuss the sperm

packaging pattern or the presence of true spermatophores (Hartnoll, 1975). Thus, studies

are required to determine if the sperm packaging patterns found in primitive crabs follows

the same model of spermatophore morphology found in Eubrachyura species. It is

expected that the spermatophores of the Dromiidae species studied here are of the

coenospermic or cleistospermic type, following the commonly described models for crabs

(El-Sherief, 1991; Guinot et al., 1997; Anilkumar et al., 1999; Klaus et al., 2009; Zara et

al., 2012; Tiseo et al., 2014, 2017).

In Brachyura, seminal fluid is transferred to females through two pairs of structures

known as gonopod one (G1) and gonopod two (G2), respectively (Hartnoll, 1969; Guinot

et al., 2013; Mclay & Becker, 2015). In Podotremata, exclusive to the Hypoconchinae and

Dromiinae subfamilies, there are also present penial tubes, which are mobile structures


59 Caunesp

formed by vas deferens protusions from gonopores on the coxa of the last thoracic

pereopod (P5) (Guinot & Tavares, 2003; Guinot et al., 2013). This sclerotized tube

receives the vas deferens contents, and it is inserted in the very long needle-like apex of the

G2, which is inserted at the base of the G1, assisting in sperm transfer (Guinot et al.,

2013). Despite being well-studied from a morphological point of view, the function of the

mobile penial tubes and the gonopods in Dromiidae during the sperm transfer is poorly

understood (Hartnoll, 1969; Guinot & Tavares 2003; Guinot et al., 2013), especially in

species with a long G2, such as Hypoconchinae and Dromiinae (McLay & Becker, 2015).

In some species of Brachyura, the end of the sperm transfer is marked by the formation of

a spermatic plug, which blocks the entrance of the vagina or vulva, preventing successive

copulations (Hartnoll, 1969; Guinot et al., 2013; McLay & Becker, 2015). Although the

absence of a sperm plug has been confirmed for Hypoconchinae, the presence of hardened

material deposited on the female sternum was found in some Dromiidae (Guinot &

Tavares, 2003; Guinot et al., 2013). However, different from the pattern found in

Eubrachyura sperm plug, presence of traces of some spermatozoa was noticed in

Dromiidae. Therefore, no studies proving the function of this secretion has carried out to

Dromiidae crabs. Due to the importance of this group for understanding the evolution of

Brachyura and the gap in knowledge of the male reproductive system of primitive crabs,

this study describes the male reproductive system of four species of Dromiidae under light

and electron microscopy. In addition, we describe the anatomy of the spermatozoa storage

structures of ovigerous females of Hypoconchinae and Dromiinae, as seen by

stereomicroscope analysis. We aim to elucidate the pattern of seminal fluid production and

chemical composition and to describe the spermatozoa package in the vas deferens and the

transfer of the seminal contents through the mobile penial tube and G2 of the male.

Moreover, we investigated the presence of the sperm plug on the spermathecal aperture of

females.

MATERIALS AND METHODS

Adult males of Hypoconcha parasitica (Linnaeus, 1763), Hypoconcha arcuata

Stimpson, 1858, Moreiradromia antillensis (Stimpson, 1858) and Dromia erythropus

(George Edwards, 1771) were collected by trawling (20 min) using a shrimp fishing boat

in the municipality of Ubatuba, São Paulo, Brazil (25°07´385´´S/47°52´508´´W), from

October of 2014 to August of 2016. The animals were transported alive in Styrofoam

boxes with proper aeration to the laboratory, where they were maintained alive in tanks.


60 Caunesp

We used at least five animals per species for all techniques, except D. erythropus, because

we collected only one specimen in two years. The animals were anesthetized by thermal

shock (-20°C/10 min), and the cephalothorax was removed. After being anesthetized, the

whole animal was fixed in 4% paraformaldehyde prepared with seawater and buffered 0.2

M sodium phosphate (pH 7.2) to view the gross anatomy of the male reproductive system

under a Leica® stereomicroscope. For histological and histochemical description, the

mature male reproductive system was removed and kept in the same fixative for 24 h

(4°C). The samples were washed twice (30 min) in the same buffer, dehydrated in an

ethanol series (70 a 95%), and embedded and enclosed in methacrylate historesin Leica®.

Serial 4-7 μm thick sections were cut on a Leica® rotary microtome and stained with

hematoxylin and eosin (Junqueira & Junqueira, 1983). For histochemistry, slides were

stained with ponceau xylidine for proteins (Mello & Vidal, 1980) and periodic acid-Schiff

(PAS) and Alcian Blue (pH 2.5) for neutral and acidic polysaccharides, respectively

(Junqueira & Junqueira, 1983). PAS combined with hematoxylin (Junqueira & Junqueira,

1983) was also used to detect the different stages of spermiogenesis. In addition, ovigerous

females of H. parasitica (N=6) and M. antillensis (N=2) from our collection preserved in

80% ethanol were also checked under a stereomicroscope to verify the structure of pleopod

one (PL1) and two (PL2) and the presence of a sperm plug on the spermathecal aperture.

For the ultrastructure analysis, fragments of AMV, MVD and PVD (1mm3) were

fixed in 2.5% glutaraldehyde in marPHEM (PHEM 1.5X+ 9% sucrose) (Montanaro,

Gruber & Leisch, 2016) for 4 h at 4°C, washed three times in PHEM buffer (Montanaro et

al., 2016) and post-fixed with 1% osmium tetroxide buffered for 2 h. The samples were

“en bloc” stained using 1% aqueous uranyl acetate (overnight), dehydrated in acetone

series, embedded and included in Epon-Araldite resin. Thin and ultrathin sections were

obtained with a Leica UC7 ultramicrotome and contrasted with 2% uranyl acetate in water

for 45 min and 0.4% lead citrate in 0.1 N NaOH for 10 min (Reynolds, 1963). The

images were obtained with a Jeol J1010 transmission electron microscope operated with an

80 kV electron beam.

The mobile penial tubes of H. parasitica, H. arcuata, M. antillensis and D.

erythropus were removed with the last thoracic pereopod (P5) The samples were fixed in

3% glutaraldehyde in sodium phosphate buffer (pH 7.2) for 24 h. All structures were

dehydrated in an ascending series of ethanol (30-100%) and completely dried in EMS 850

critical-point using liquid CO2. The materials were placed on SEM stubs and sputter-

coated with gold (5 nm) with Dayton vacuum sputtering. The samples were observed and


61 Caunesp

photographed in a scanning electron microscope (JEOL JSM5410) using a 15 kV electron

beam.

To describe the mechanism by which seminal fluid packs the spermatozoa, 2 cm-

long fragments of vas deferens of H. parasitica and M. antillensis were squeezed for

luminal content extrusion using forceps to release the seminal fluid onto slides containing

seawater. In addition, some material found on the female spermathecal aperture of both

species were removed onto slides and stained with neutral red to check for the presence of

spermatozoa. As the female M. antillensis was fixed in 100% alcohol, we used a

spermatozoon fixed in 4% paraformaldehyde prepared with seawater and 0.2 M sodium

phosphate buffered to compare the two morphologies. All samples were analyzed under a

differential interference phase contrast microscope (Zeiss Axio Scope Z2). To verify the

presence of a sperm plug and to describe the morphology of the paired female pleopods in

Hypoconchinae and Dromiinae, females of H. parasitica (n=5) and M. antillensis (n=5)

were obtained from the collection of the Invertebrate Morphology Laboratory (UNESP -

Jaboticabal). The dorsal external structures were examined under a Leica®

stereomicroscope and photographed using the Leica IM50 program.

RESULTS

GROSS ANATOMY

In H. parasitica, H. arcuata, M. antillensis and D. erythropus, the male reproductive

system is a bilateral “H”- shaped organ composed of a pair of testes, located in both the

superior cephalothorax sides, without reaching the lateral margins (Fig. 1A - C). Each pair

of testes is a whitish and convoluted tubular organ. Connected to the testes is a pair of vasa

deferentia, which extend longitudinally over the hepatopancreas and below the heart,

toward the ventral posterior region of the body (Fig. 1A - C). Each vas deferens ends in the

ejaculatory duct that opens in the mobile penial tube. The vas deferens has no different

anatomical regions or accessory glands. The vasa deferentia are convoluted; however, in

Hypoconchinae they are left-right folded (Fig 1A), while in the Dromiinae, M. antillensis

and D. erythropus (Fig. 1B, C) are back and forth (posterior-anterior) convoluted (Fig.1B,

C). In D. erythropus, the posterior region of the vas deferens is quite convoluted (Fig. 1C).


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HISTOLOGY AND HISTOCHEMISTRY

TESTIS

The testes of H. parasitica, H. arcuata, M. antillensis and D. erythropus showed

similar features both macroscopically and histologically. The testes were classified as

tubular with a histological analysis, and they are internally filled by germ cells in different

stages of spermatogenesis and spermiogenesis (Fig. 2A - D). Spermatogenesis starts in the

germinal zone, which is composed of many spermatogonia localized at the periphery of a

seminiferous tubule (Fig. 2E). The spermatogonia are differentiated by a large central

nucleus, several nucleoli and an acidophilic cytoplasm (Fig. 2E). In all Dromiidae sections

analyzed, the maturation zone beneath the germinal center only showed spermatocytes or

spermatids in different maturation stages (Fig. 2F - J). The maturation zone was not found

to contain different cell strata since spermatocytes until late spermatids in the same cross-

section. The chromosomes in different stages of the meiotic prophase characterize the

primary spermatocytes (Fig. 2F). The secondary spermatocytes have smaller nuclei, which

are rounded and filled with homogeneously stained chromatin (Fig. 2G).

The spermiogenesis showed a similar pattern of cell maturation in all studied species

by light microscopy. Early spermatids are identified by the appearance of the proacrosomal

vesicle, which is reactive with homogeneous staining to PAS (Fig. 2H). The nucleus is

rounded, homogeneously basophilic and occupies a large part of the cell volume (Fig. 2H).

The intermediary spermatids are marked by the beginning of the nuclear modification

becoming slightly irregular due to an increase in the round acrosome vesicle (Fig. 2I). The

main characteristic of this cell is the presence of the PAS-positive acrosome exhibiting a

more intense reaction at the perforatorial chamber and the acrosomal operculum, both

during formation (Fig. 2I). Late spermatids contain thin “half-moon” nuclei that became

discoid, positioned in the opposite pole of the acrosome vesicle (Fig. 2J). The nucleus has a

cup appearance, almost surrounding the acrosome (Fig. 2J). Mature spermatozoa are

similar to the late spermatids; however, they are found in the seminiferous tubule lumen

(evacuation zone) (Fig. 2K). This cell shows a slightly thinner nucleus, which almost

completely surrounds the heterogeneous acrosomal vesicle (Fig. 2K). The evacuation zone,

or seminiferous tubule, of H. parasitica, H. arcuata and M. antillensis is filled with only

one basophilic secretion, called a type I secretion (Fig. 2K). However, in D. erythropus, the

type 1 secretion contains a lucid secretion that resembles bobbles near the periphery of the


63 Caunesp

evacuation zone, which spreads on a basophilic secretion where the spermatozoa are

immersed (Fig. 2L).

VAS DEFERENS

Despite the absence of different anatomical and histological regions along the vas

deferens or the presence of accessory glands, the species studied here differed in their

seminal fluid secretions and chemical compositions as follows:

Hypoconcha parasitica. - The mature spermatozoa are conducted into the vas deferens

immersed in type I secretion, the same type found in the testes (Fig. 3A). The vas deferens

lumen exhibits, in the anterior region, spermatozoa in a type I secretion surrounded by type

II secretion (Fig. 3A - C). The type II secretion is electron-dense and homogeneous, while

the type I secretion is less electron-dense and finely granular (Fig. 3B). The volume of the

type II secretion is smaller in regions near the testis (Fig. 3C), becoming larger along the

vas deferens (Fig. 3D - G). The vas deferens did not show histological variation or

different cell types in the anterior, middle or posterior regions. It has a simple epithelium,

lying on a thick musculature surrounded by a thin layer of connective tissue (Fig. 3A). The

epithelium along the vas deferens ranged from cubic to flattened due to the presence of a

greater amount of type II secretions (Fig. 3A, C). The spermatozoa are compacted into

large central masses in the type I secretion, forming a large spermatic cord (Fig. 3A - G).

This cord is convoluted within the type II secretion in some places (Fig. 3G). A cross-

section confirms the single, continuous tubular morphology of the vas deferens along its

whole extension (Fig. 3C). The type I secretion is acidophilic (Fig. 3D) and reactive to

proteins (Fig. 3E), being positive for neutral (Fig. 3F) and acidic polysaccharides (Fig.

3G). The type II secretion acts as a wrap around the spermatic cord, producing a unique

extremely elongated kind of coenospermic “spermatophore”. This type II secretion is

basophilic (Fig. 3D), intensely stained to proteins (Fig. 3E) and to neutral polysaccharides

(Fig. 3F), being weakly positive for acidic polysaccharides (Fig. 3G). Inside of the type II

secretion, fibrous and acidophilic glycoprotein materials are present, along with strongly

marked neutral polysaccharides (Fig. 3D - F). The basophilic spermatozoa are strongly

reactive for proteins and positive for neutral and acid polysaccharides (Fig 3D - G).

Hypoconcha arcuata. – The mature spermatozoa in the vas deferens are also immersed in

type I secretions (Fig. 4A). From the anterior to the posterior region of the vas deferens,

the large spermatic mass is immersed in type I secretion, forming a cord surrounded by

type II secretion (Fig. 4A - C). In a cross-section, the spermatozoa form a central mass


64 Caunesp

covered by the electron-dense and granular type I secretions, and the mass is surrounded

by type II secretion, which is homogeneously electron-dense and basophilic (Fig. 4B, C).

The internal sperm mass remains as a long spermatic cord surrounded by the type II

secretion (Fig 4A, C). The vasa deferentia in all regions are formed by a simple epithelium,

surrounded by musculature and a thin layer of connective tissue. In this species, the

musculature is thicker than in the previous one, showing several layers (Fig. 4D). From a

histochemical point of view, the type I secretion is weakly basophilic (Fig. 4D), slightly

reactive for proteins (Fig. 4E) and neutral polysaccharides (Fig. 4F), but no acidic

polysaccharides were detected (Fig. 4G). The type II secretion is acidophilic (Fig. 4D),

weakly reactive for proteins (Fig. 4E) and neutral polysaccharides (Fig. 4F) and not

reactive to acidic polysaccharides (Fig. 4G). The basophilic spermatozoa are strongly

proteinaceous and also reactive to neutral and acidic polysaccharides (Fig. 4A, C - G).

Moreiradromia antillensis. – Mature spermatozoa in the vas deferens are immersed in the

type I secretion, adjoin this from the anterior to the posterior region, and have an outer

layer of type II secretion. The type II secretion contains many spheres or granules of

different sizes immersed in a finely granular matrix (Fig. 5A - C). The type I secretion is

electron-dense and homogeneous, while the type II is a finely granular matrix and is less

electron-dense than the type I (Fig. 5B). The granules are electron-dense (Fig. 5B). The vas

deferens is formed by a simple cubic epithelium on a thin layer of connective tissue,

surrounded by strong musculature (Fig. 5A, D). Spermatozoa in the type I secretion form a

long spermatic cord within the type II secretion (Fig. 5A). In cross-section, the vas

deferens is a spermatic cord arranged centrally, surrounded by type II secretion (Fig. 5C).

The type I secretion is basophilic (Fig. 5A, C, D), weakly reactive to proteins (Fig. 5E),

and neutral (Fig. 5F) and acidic polysaccharides (Fig. 5G). Type II secretions have

granules strongly reactive to proteins (Fig. 5E) and negative to polysaccharides (Fig. 5F,

G). The type II secretion matrix is finely granular and positive for proteins (Fig. 5E) and

neutral polysaccharides (Fig. 5F) but is weakly responsive to acidic ones (Fig. 5G). The

basophilic spermatozoa are strongly reactive to all compounds studied (Fig. 5A, C - G).

Dromia erythropus. – This species showed a higher degree of complexity in the chemical

composition of secretions and sperm packaging. Mature spermatozoa are conducted

through the vas deferens immersed in type I secretion, which is electron-dense and

homogeneous, forming the spermatic cord (Fig. 6A, B). The vas deferens lumen has a

spermatic cord beginning from the anterior region of the vas deferens, surrounded by type

II, III and IV secretions (Fig. 6A - C). The epithelium along the vas deferens varies from


65 Caunesp

cubic to squamous, probably due to the presence of varying amounts of type II secretions

in the vas deferens, independent of the region (Fig. 6A, C, D). The spermatic cord is

compacted by type II secretion, which contains lucid spherical granules under the light

microscope; these granules are electron-dense under TEM (Fig. 6A, B). The matrix

between the granules shows the same electron density as is found in the type I secretion of

the spermatic cord (Fig. 6B). The type I secretion is weakly basophilic and

glycoproteinaceous with the presence of only neutral polysaccharides (Fig. 6D - F). The

type II secretion is granular and strongly proteinaceous (Fig. 6E) while the type III is

electron-dense with discrete electron-lucid granules (Fig. 6B). This secretion seems more

fluid, weakly fibrillar and basophilic and is negative for proteins and weakly reactive to

neutral and acidic polysaccharides (Fig. 6D - G). The type IV secretion is heterogeneous

and composed of two elements. One is more abundant on the face in contact with the type

III secretion, which is less electron-dense (Fig. 6B), basophilic, and weakly reactive to

proteins but strongly reactive to neutral polysaccharides and slightly reactive to acidic

polysaccharides (Fig. 6D - G). The other element is next to the epithelium and is electron-

dense, acidophilic, glycoproteinaceous, and positive for neutral polysaccharides (Fig. 6B –

F). However, for acidic polysaccharides it is less intense and negative (Fig. 6G). The type

IV secretion is thinner in regions near the testis (Fig. 6C), becoming thicker along the vas

deferens (Fig. 6A, C - G). The basophilic spermatozoa are strongly reactive to proteins and

neutral polysaccharides and reactive to acidic polysaccharides (Fig. 6D – G). The summary

of the results from the histochemical reactions of the spermatozoa and the different types

of secretions in the vas deferens for the four species of Dromiidae are shown in Table 1.

ULTRASTRUCTURE OF THE VAS DEFERENS

As the four species of Dromiidae vasa deferentia have the same cell ultrastructure,

we will use H. parasitica as a model for the description. The vas deferens wall is

composed of an external connective layer, a middle muscular layer and an internal

epithelium (Fig. 7A). The lumen is filled by a less electron-dense secretion where the

spermatozoa are distributed and other secretions that show different electron densities

depending on the species. No spermatophore wall is observed around the spermatic cord

(Fig. 7A). The thin connective tissue layer contains fibroblast-like cells with oval or flat

nuclei (Fig. 7A, B). The cytoplasm contains polyribosomes, rough endoplasmic reticulum

(RER) and some mitochondria (Fig. 7B). More than one layer makes up the striated

muscular fibers, showing at least one longitudinal fiber and other oblique fibers (Fig. 7A,


66 Caunesp

B). Epithelial cells have a thick basal lamina, which is filled by different electron densities

(Fig. 7B, C). The basal region of the epithelial cell depicts many deep basal plasma

membrane folds and many mitochondria and polyribosomes are noticeable among them

(Fig. 7C). The cytoplasm is filled with a large amount of RER, and the nucleus is

positioned in the middle of the cell (Fig. 7D). The nucleus is usually flattened and contains

more condensed chromatin near the nuclear envelope (Fig. 7A, D). The RER shows a

traditional pattern composed of many parallel cisternae (Fig. 7E), and among them, many

mitochondria and well-developed Golgi complexes are noticeable (Fig. 7E, F). The Golgi

complex produces at least two types of vesicles, one electron lucent and another filled with

granular and electron-dense material; both are released into the lumen by exocytose at the

apical region (Fig. 7G, H). The epithelium contains small microvilli regularly distributed

on the surface (Fig. 7G, H).

MOBILE PENIAL TUBE AND GONOPOD

The penial tube is a long conical-round paired organ, forming a mobile tube

independent of the coxal structure, emerging from male P5 in all studied species (Fig. 8A -

D). The scanning electron microscope shows that this structure in the Hypoconchinae H.

parasitica and H. arcuata is composed of setae, mainly on the basal portion (Fig. 8A, B).

One of the faces does not contain setae, forming a glabra margin (Fig. 8A, B). The setae

are long and show many ramifications classified as plumose (Fig. 8A, B). In the Dromiinae

M. antillensis and D. erythropus, the penial tube is long, flat dorso-ventrally, and slightly

curved at the apex (Fig 8C, D). The basal setae surround the penial tube, and a line of setae

occurs along the central margin 2/3 (Fig 8C, D). The setae are long in both the latter

species and were classified as pappose types (Fig 8C, D). In all species, the penial tube

apex has a very thin cuticle, noticeable by the wrinkles caused by dehydration, which

forms a mobile operculum (Fig 8E - H). In the Dromiidae species studied here, the penial

tube is inserted into the gonopod G2, which shows a needle-like apex and so easily

fractures (Fig. 8I). The fractured G2 in M. antillensis contains the secretion that forms a

smooth surface around the spermatozoa. However, the secretion is thinner under MEV,

also due to the dehydration process (Fig 8I, J). The same pattern was observed in the other

Dromiidae species.


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DIFFERENTIAL INTERFERENCE CONTRAST PHASE MICROSCOPE (DIC)

The seminal fluid, when mechanically squeezed out of the vas deferens, maintains its

structure, forming a central spermatic cord surrounded by secretions differing according to

species. In the Hypoconchinae H. parasitica, the secretion around the spermatic cord is

thin and homogeneous under DIC (Fig. 9A). A similar result was found in Dromiinae (Fig.

9B). In M. antillensis, the mechanically squeezed seminal fluid also formed an elongated

cord composed of an outer rounded type II secretion and inner spermatozoa contained in

type I secretion (Fig. 9B). This pattern of an elongated and unique coenospermic

“spermatophore” is found in all Dromiidae species studied.

“SPERM PLUG” AND FIRST FEMALE PLEOPOD

The pair of uniramous first pleopods (PL1) is held in the medial portion of the

spermathecae in H. parasitica, almost perpendicular to the body axis but not reaching the

spermathecal apertures (Fig. 10A). Their lateral edge apices are fringed with setae (Fig.

10A, B). The pair of spermathecae is formed by the split of the intersegmental phragma

seven and eight sternites, and the width brings them almost near the third coxae, where the

gonopores are located (Fig. 10A). In ovigerous females of H. parasitica, material similar to

a sperm plug on the spermathecal aperture was found and this material included traces of

spermatozoa (Fig. 10A, C, D). In M. antillensis, the pair of uniramous PL1 is present in the

1/3 portion of the spermathecae, not reaching the spermathecal aperture (Fig. 10E). Their

edge apices are also fringed with setae (Fig. 10E, F). The PL1 of ovigerous females of both

subfamilies did not seem to be involved in carrying eggs once the PL1 is free, while the

other pleopods held eggs. In the ovigerous females of M. antillensis, material similar to a

sperm plug was also found on the aperture of spermathecae with traces of spermatozoa

(Fig. 10G, H, I). The spermatozoa found in the sperm plug of M. antillensis were fixed in

100% alcohol and show a similar morphology compared to the sperm fixed in 4%

paraformaldehyde prepared with seawater and 0.2 M sodium phosphate buffer (Fig. 10I, J).

DISCUSSION

The male reproductive system of Dromiidae showed significant differences from

previously described Podotremata and especially with Eubrachyura. Based on observations

of the male reproductive anatomy, no different vas deferens regions or accessory glands,

caeca or lateral out pockets were found, which is distinct from the pattern described for the

Podotremata Ranina ranina (Linnaeus, 1758) by Minagawa et al. (1994), Dromia


68 Caunesp

personata by Hartnoll (1975) and all eubrachyurans studied (Hartnoll, 1964; Beninger et

al., 1988; Krol et al., 1992; Garcia & Silva 2006; Castilho et al. 2008; Simeó et al., 2009;

Stewart et al., 2010; Nicolau et al., 2012; López Greco, 2013; Ravi, Manisseri & Sanil,

2014; Tiseo et al., 2014; McLay & Becker, 2015). Small differences were noticed between

the reproductive system subfamilies since the reproductive systems in the Dromiinae (M.

antillensis and D. erythropus) are longer and convoluted, mainly in the distal portion of the

vas deferens, compared to Hypoconchinae. In R. ranina, there are three anatomically

recognized regions (anterior, middle and posterior) without any lateral expansions, caeca

or accessory glands. However, the anterior portion is more modified, thin and convoluted

(Minagawa et al., 1994). Despite the anatomy of D. personata not yet being described,

there seems to be some differences in histology that may imply anatomical differences,

though without any glands (Hartnoll, 1975). Thus, the male reproductive system in

dromiids is extremely simplified and different from the patterns described for the other

primitive crabs.

The Dromiidae testis was classified as tubular based on Nagao & Munehara (2003).

The testes show different areas with different kinds of spermatogenesis cells, but all stages

were never observed in the same transversal section, as observed in Maja brachydactyla,

Balss, 1922 by Simeó et al. (2009). As described in this latter species and also in

Pachygrapsus, Randall, 1840, the mature sperm cells are released to the evacuation zone

(lumen of seminiferous tubule). Spermatogenesis in Dromiidae also shows the same

histological characteristics found in other species, which seems to be the general model in

Brachyura crabs (Ryan 1967; Garcia & Silva 2006; Castilho et al., 2008; Santos et al.,

2009; Zara et al., 2012; Tiseo et al., 2014). Additionally, the classification of early, middle

and late spermatids, based on the increase of the acrosomal vesicle, also follows the pattern

found in Eubrachyuran crabs under light microscopy (Zara et al., 2012; Nascimento &

Zara, 2013; Tiseo et al., 2014). However, different from these authors, in the present study,

the cell maturation states were easily identified by the use of PAS with hematoxylin

techniques instead of toluidine blue stain. Therefore, we propose that the use of this

technique is more appropriate for the description of spermiogenesis in Brachyura.

The vas deferens of Dromiidae is a single tubule, simple and continuous, and any

significant morphological variation under light and electron microscopy is in agreement

with anatomy. This pattern of vas deferens, without clear regions or glands, is unique

among Brachyuran crabs. In Podotremata, the early descriptions of R. ranina report

differentiated regions, including the presence of epithelium and secretions that were absent


69 Caunesp

in all species in the present work. However, despite being described as different regions by

the characterization of the epithelium and the nature of the secretions, D. personata and

Homola barbata (Fabricius, 1793) are discussed as having no gross morphology

specialization (Hartnoll 1975). Thus, based on our studied species, we corroborate the

results of D. personata, and this morphology can be considered the simplest model in

Brachyura. Moreover, the simplified pattern of the vas deferens of Dromiidae is different

from that described for shrimps in Dendrobranchiata and Caridea, where the vas deferens

is differentiated into proximal, middle and distal regions, and also contains terminal

ampoule (Bauer, 1986; Bauer & Martin, 1990; Chow et al., 1991; Bauer & Min, 1993;

Bauer, 2004). However, the absence of distinct regions in the vas deferens is a commonly

observed pattern in Anomura (Buranelli, Zara & Mantelatto, 2014) and some Astacidea

(Kooda-Cisco & Talbot, 1982; Dudenhausaennd & Talbot, 1983; López Greco et al., 2007;

López Greco, 2013). In these infraorders, although it was possible to divide these tubules

into different regions, what was normally observed was the increase of the diameter of the

posterior or distal region of the vas deferens.

The vas deferens wall in Dromiidae was divided into three layers: an outer thin

connective layer, a middle muscular layer and an inner epithelium; this may be considered

a pattern found in all decapods studied under TEM (Hinsch & Walker, 1974; Kooda-Cisco

& Talbot, 1986; Ro et al., 1990; Benhalima & Moriyasu, 2000; Simeó et al., 2009). The

Dromiidae muscular layer is thick, and the fibers are longitudinal and oblique along the vas

deferens, indicating that they have the same function throughout the tube and during sperm

transfer. Usually, the muscular layers in Decapoda show different fiber orientations and

may be more or less developed according to the vas deferens region associated with the

role of each portion (Kooda-Cisco & Talbot, 1986; Ro et al., 1990; Benhalima &

Moriyasu, 2000; Simeó et al., 2009). For example, in the AVD and MVD, the musculature

is related to spermatophore formation and movement to the PVD, where the fibers have a

role in mixing the spermatophores with the accessory gland fluid, as well as in aiding

sperm transfer (Ryan, 1967; Simeó et al., 2009; Tiseo et al., 2014). The cells from the

epithelial layer along the vas deferens in Dromiidae show the same pattern as merocrine

glycoprotein secretory cells. These cells are characterized by their basal membrane which

is folded with many mitochondria among them, and this is likely related to the high ionic

exchange in the secretory regions of crab vas deferens (Hinsch & Walker, 1974; Simeó et

al., 2009), indicating an active uptake of compounds from the hemolymph, as has been

suggested for insect salivary glands (Zara & Caetano, 2002). In addition, the cytoplasm is


70 Caunesp

filled with a large amount of RER and large, well-developed Golgi complexes, producing

small secretory vesicles that are released by exocytosis, corroborating the histochemical

results. This cell ultrastructure, including the small number of secretory vesicles, is similar

to what is found in spider crabs and others Decapoda (Hinsch & Walker, 1974; Kooda‐

Cisco & Talbot, 1986; Ro et al., 1990; Simeó et al., 2009).

The spermatozoa packaging in the four dromiid species is different from what has

been typically reported for crabs in general. These species exhibit a long spermatic cord,

surrounded by glycoproteins, forming a kind of very elongated coenospermic

“spermatophore”, without wall or capsule. This pattern is different from all others

described for Eubrachyura. Spherical or elliptical spermatophores of the coenospermic

type is the most common structure observed for Heterotremata and Thoracotremata (for

review, see Erkan et al., 2009; Zara et al., 2012; Tiseo et al., 2014, 2017). In the same

groups of crabs, cleistospermic spermatophores are uncommon; however, they are found in

some freshwater crabs, such as Potamidae, Gecarcinucidae and a single marine crab

Pachygrapsus gracilis (Saussure, 1858) (Guinot et al., 1997, Klaus et al., 2009; Klaus &

Brandis, 2011; Tiseo et al., 2014, 2017). In Podotremata, the only spermatophores

described appear to be similar to those of the Dromiidae studied here, despite the

phylogenetic distance between the Dromioidea and Raninoidea clades (Tsang et al., 2014).

The spermatophore in R. ranina is composed of a central mass of cylindrical spermatozoa

surrounded by two secretions that make up the wall, named the “capsule” (Minagawa et

al., 1994). However, these authors do not discuss the role of the capsule or whether this

structure can be considered a true spermatophore or not. Minagawa et al. (1994) did not

show whether this spermatophore is a single structure with a spermatic cord inside, as was

found in the species studied here. The only description of R. ranina reported the

spermatophore as small, deposited near the exit of the spermathecae, and other authors

have proposed that larger part of the spermatophore is internalized in the spermathecal,

without due verification (Minagawa, 1993; Minagawa et al., 1994). In D. personata, the

vas deferens was divided into three regions according to the epithelial cells and luminal

content. The spermatozoa are maintained centrally, agglutinated in a secretion along the

vas deferens, with a second layer of vacuolar secretion added at the MVD and a third

secretion added at the PVD, where no spermatozoa are present (Hartnoll, 1975;

Subramoniam, 1993). These luminal secretions are very similar to those described in D.

erythropus; however, in this species the luminal secretion is the same from the AVD to the

PVD, which also contain spermatozoa. The male reproductive system anatomy and


71 Caunesp

histology of Hypoconchinae and the Homolidae Ho. barbata are quite similar, including

the presence of only S1 and S2 in the seminal fluid. However, no epithelial cell differences

were found in Hypoconchinae as they occur in Ho. barbata (Hartnoll, 1975).

Thus, the Dromiidae secretion types in the vas deferens and the spermatic cord,

forming the long coenospermic spermatophores, are more similar in structure to D.

personata and Ho. barbata than R. ranina. These similarities are in agreement with

molecular phylogenetic studies by Ahyong et al. (2007) and Tsang et al. (2014) using

nuclear protein-coding and mitochondrial rRNA genes, which showed that Dromiidae and

Homolidae are more closely related than Raninidae. Tsang et al. (2014) recognized

Podotremata in separate sections: Dromiacea, Raninoidea and Cyclodorippoidea.

Dromiidae and Homolidae are inside Dromiacea, which is in accordance with the male

reproductive system morphology reported in this study and previous reports (Hartnoll,

1975; Guinot, 1977; Minagawa, 1993; Minagawa et al., 1994).

The Dromiidae have already been included in the Anomura infraorder (Spears, Abele

& Kim 1992). However, the sperm packaging in Dromiidae is clearly different from all

Anomura spermatophores described, although the anatomy of the vas deferens is similar.

In Anomura, the spermatozoa are included in pedunculate coenospermic spermatophores,

which are composed of an ampulla, a peduncle and base or foot (Tudge, 1991; Scelzo,

Mantelatto & Tudge, 2004; Amadio & Mantelatto, 2009; Buranelli & Mantelatto, 2012;

Buranelli et al., 2014). In Astacidea, sperm masses form an elongated cord and are

surrounded by different types of secretions in the vas deferens lumen, which seems to be a

convergence with the species of this study. These secretions in lobsters are added from the

anterior region of the vas deferens, with new layers added to the wall in the middle and

posterior regions thus making them thicker (Hobbs et al., 2007, López Greco, 2013). This

multilayer wall structure is called a tubular spermatophore, which is elongated and occurs

in Parastacidae, Enoplometopidae, Nephropidae, Homaridae, Astacidae and Cambaridae

(Kooda-Cisco & Talbot, 1982; Dudenhausen & Talbot; 1983; Hobbs et al., 2007; López

Greco et al., 2007; López Greco, 2013). However, the spermatophores in H. parasitica, H.

arcuata, M. antillensis and D. erythropus do not show these layers on the wall. Therefore,

they are similar but not identical to the spermatophores found in Astacidea (Kooda-Cisco

& Talbot, 1982; Dudenhausen & Talbot; 1983; Hobbs et al., 2007; López Greco et al.,

2007; López Greco, 2013). Thus, the present results strongly agree with the early

proposition of Hartnoll (1975) that the long coenospermic spermatophores of D. personata


72 Caunesp

and the four species of Dromiidae in this analysis are more similar to Astacidae than

Eubrachyura.

To insert the elongated spermatophores in the narrow spermathecae, the dromiid

species use a very long, thin gonopore G2 attached to the gonopore G1. At the base of G2,

the long mobile penial tube of the studied Dromiidae species emerges from P5 coxa,

corroborating previous anatomical studies of Dromiinae and Hypoconchinae subfamilies

(Guinot & Tavares, 2003; Guinot & Quenette, 2005; Guinot et al., 2013). According to

Guinot & Tavares (2003), the penial tube is the vas deferens prolongation to the external

environment, forming a sclerotized tube with a soft tip, and is only characteristic of

subfamilies Hypoconchinae and Dromiinae. Our results confirm the soft tip, and we

propose that the soft tip acts as an operculum, allowing the seminal fluid and the spermatic

cord to pass in one direction, avoiding backflow during the copular pumping. The penial

tube and the G2, including the operculum described her, enters into a single foramen in

male gonopod G1. Additionally, the G2 with a needle-like apex may submit the high

pressure the elongated spermatophores, during sperm transfer. Thus, this penial tube (and

the operculum), as already stated, probably plays an important role in the reproductive

strategy of these species (Guinot & Quenette, 2005). Moreover, we observe that during the

spermatic transfer of Hypoconchinae, the male abdomen performs a movement of

extension and distension, which seems to collaborate with the propulsion force to transfer

the elongated spermatophore (Bento-Garcia MA & Zara FJ., unpublished data). In

Eubrachyura, some species of Dorippidae have a long penial tube, similar to the mobile

tube and independent from the coxa found in Dromiidae. However, the dorippid penial

tube is coxo-sternal and is not completely free from the coxa (Guinot & Tavares, 2003;

Guinot et al., 2013; Hayer et al., 2016; Becker & Scholtz, 2017), probably functioning in a

different manner than dromiids. In Dorippidae, the seminal receptacle is also completely or

almost completely cuticle-lined, very similar to the Dromiidae spermathecae (Hayer et al.,

2016; Becker & Scholtz, 2017; Vehof, Scholtz & Becker, 2017). However, in doripids, the

ovary and oviduct are in contact with the vagina or vulva where internal fertilization takes

place (Hayer et al., 2016; Vehof et al., 2017). In Podotremata, including Homolidae and

Dromiidae, the spermathecae has no internal connection to the oviduct, leading to an

external fertilization (Guinot & Quenette, 2005; Guinot et al., 2013; Becker & Scholtz,

2017). In this case, the seminal receptacle of dorippids and the spermathecae of dromiids

seem analogous instead of homologous since the positions on the body are also distinct


73 Caunesp

(i.e., sternite 6 in Dorippidae and sternite 7 and 8 in Dromiidae) (Guinot & Quenette, 2005;

Guinot et al., 2013; Becker & Scholtz, 2017).

The spermatozoa or spermatophores stored in the spermathecae are poorly studied in

Podotremata, especially in dromiids. Although the absence of “sperm plugs” in

Hypoconchinae was earlier stated (Guinot & Tavares, 2003) and we also confirmed this

absence in H. parasitica and H arcuata. The presence of sperm plug secretion was

observed in the Dromiinae D. personata (Hartnoll, 1975), Austrodromidia octodentata

(Haswell, 1882) and Pseudodromia latens Stimpson, 1858 and is described as a hardened

material deposited on the female sternum (Guinot & Tavares, 2003; Guinot et al., 2013). It

was also observed in Lauridromia intermedia (Laurie, 1906), with traces of spermatozoa

on this plug material (Guinot & Quenette, 2005). In our study, we found this material

deposited on the aperture of spermathecae in M. antillensis ovigerous females, also

including traces of spermatozoa, following the descriptions of Dromiinae (Guinot &

Tavares, 2003; Guinot & Quenette, 2005; Guinot et al., 2013). According to Guinot &

Tavares (2003) and Guinot et al. (2013), this secretion is a sperm plug, acting as a rigid

adhesive secretion on the spermathecal aperture in Podotremata. The sperm plug (internal

or external) in brachyurans is considered a barrier that prevents successive copulations,

among other functions (for review, see Hartnoll, 1969; Zara et al., 2012; Zara, Raggi

Pereira & Sant’Anna, 2014). If this secretion founded in Dromiinae act as a sperm plug it

shows the same function observed in Eubrachyura, however, the main composition is

sliglithly different since eubrachyurans sperm plug lacks the presence of spermatozoa. In

addition, we also founded a presence of secretion in H. parasitica apertures and it was only

observed in ovigerous females. Thus, further studies need to be carried out to demonstrate

if this secretion is a sperm plug in Hypoconchinae or if this secretion is an evidence of

material sternalized from spermatheca during the external fertilization in Dromiidae crabs.

McLay & Becker (2015) proposed the use of “sperm plaque” in Podotremata because

this secretion is not necessarily injected in the spermathecae. This structure is more similar

to a "barrier" that can prevent the loss of spermatozoa and the influx of water and also

avoids subsequent copulations in the spermatic competition (McLay & Becker, 2015;

Becker & Scholtz, 2017). However, based on the patterns of seminal fluid production and

sperm packaging in all species studied here, and based on the presence of spermatozoa in

the secretions on the spermathecal aperture of ovigerous H. parasitica and M. antillensis

females, the sperm plug function seems questionable to us. Although mating has been

described once for Dromiidae and Podotremata (Hartnoll, 1975; Guinot et al., 2013), we


74 Caunesp

propose two hypotheses: 1) The long coenospermic spermatophores formed by seminal

fluid and the inner spermatic cord are inserted inside the spermathecae and some of the

seminal fluid and seminal cord are released from the spermathecae on the aperture of

spermathecae as a result of the copulation; 2) As the fertilization in Podotremata occurs

outside the body, the spermatozoa and seminal fluid are released from the storage chamber

(spermathecae) to meet the extruded eggs during the ovulation process. Thus, the material

found outside the spermathecal aperture can be remnants of spermatozoa and seminal fluid

extruded by the female during ovulation. The production of the glycoprotein secretion S1

acts as an energetic resource to maintain viable spermatozoa as proposed in many

Eubrachyura (Zara et al., 2012, 2014), while the other external layers with acidic and

neutral polysaccharides may act as an adhesive secretion, attaching the material in the

spermathecae and also avoiding loss of spermatozoa. This hypothesis was proposed when

the vas deferens was squeezed and observed under DIC microscopy, where the

spermatozoa remained enveloped by a secretion, maintaining its long constitution. This last

observation consolidated the proposal that the secretion continues to surround the

spermatozoa after ejaculation and until the fertilization. Hartnoll (1975) also described the

spermatozoa surrounded by several types of secretions inside the spermathecae of D.

personata, strengthening our hypothesis. On the other hand, in species of Homoloidea, free

spermatozoa were found mixed with seminal fluid inside the spermathecae, without the

presence of a sperm plug (Hartnoll 1975, Becker & Scholtz, 2017). Hartnoll (1975)

reported that the “plug secretion” appears after the copulation, and the acid

polysaccharides found in the external layers can also promote the extravasations of

material from the inseminated spermathecae. The acidic polysaccharides can attract

sodium and water from the seawater, leading to the hydration of the S1 and S2 secretions

in M. antillensis, as well the S3 secretion in D. erythropus, producing the expansion of

these secretions inside the spermathecae, leading to the formation of the plug. Since

Hypoconcha secretions are weakly responsive to acidic polysaccharides, the hydration

process probably would be not so effective to release material on the spermathecal

aperture.

The spermathecae in Homolidae are very similar to the Dromiidae and also show

muscles attached to the inner wall (Hartnoll, 1975; Becker & Scholtz, 2017). The females

of Hypoconcha and Moreiradromia have a similar structure of the PL1 as observed in the

Homolidae H. barbata and H. orientalis, Henderson, 1888, by Becker & Scholtz (2017).

Furthermore, the PL1 in the observed Dromiidae ovigerous females did not seem to be


75 Caunesp

destined to carry eggs once the PL1 was free, while the other pleopods held eggs. In

Homolidae, the PL1 seems to draw extruded eggs into the fertilization chamber (Becker &

Scholtz, 2017). When the eggs are draws the PL1 are held erect so that the eggs remain in

this chamber until the sperm are released for fertilization; afterwards, the PL1 return to

their initial position to fertilize eggs attained by PL2- PL5 in the brood chamber (Becker &

Scholtz, 2017). This mechanism has been described in American Lobster Homarus

americanus Milne Edwards, 1837(Aiken, Waddy & Mercer, 2004), and although the PL1

do not cover the spermathecal apertures, they probably play a similar role in the Dromiid

species studied here.

In conclusion, this research provides descriptions of a different pattern of male

reproductive system that can be considered characteristic of the primitive Brachyura. The

vas deferens showed no anatomical, histological and cellular variations from the anterior to

the posterior region. Additionally, the spermatozoa package on the spermatic cord, forming

an elongated spermatophore, can be considered unique and is probably the simplest and

most basal coenospermic spermatophore described for Eubrachyura, similar to the external

fertilization of species of Astacidae. This pattern seems to be exclusive to Podotremata (at

least Dromiidae and Homolidae), and no direct evidence of intermediary steps can link

Podotremata and Eubrachyura, in the same way to the presence of function sperm plug in

Dromiidae.

ACKNOWLEDGMENTS

FJZ and MAGB thank the São Paulo Research Foundation (FAPESP grants BIOTA

#2010/50188-8, IC #2014/21294-5, MS #2016/10394-4). FJZ also thanks the

Coordination for the Improvement of Higher Education Personnel (CAPES), Ciências do

Mar II #1989/2014. The authors are also grateful to Marcia F. Mataqueiro for technical

support and the Electron Microscopy Laboratory of FCAV, UNESP – Campus of

Jaboticabal facility. We also thank the fisherman Djalma Rosa for help during the

collection of these rare animals. This study was conducted in accordance with Brazilian

laws (FJZ - MMA SisBio Licence #34587-1).

REFERENCES

Ahyoung TS, Lai JCY; Sharkey D, Colgan DJ, Ng PKL. 2007. Phylogenetics of the

brachyuran crabs (Crustacea: Decapoda): The status of Podotremata based on small

subunit nuclear ribosomal RNA. Molecular Phylogenetics and Evolution 45:576-

586.


76 Caunesp

Aiken DE, Waddy SL, Mercer SM. 2004. Confirmation of external fertilization in the

American lobster, Homarus americanus. Journal of Crustacean Biology 24: 474-480.

Amadio LM, Mantelatto FL. 2009. Description of the male reproductive system of the

hermit crab Calcinus tibicen (Decapoda: Anomura: Diogenidae). Journal of

Crustacean Biology 29:466-475.

Anilkumar G, Sudha K, Subramoniam T. 1999. Spermatophore Transfer and Sperm

Structure in the Brachyuran Crab Metopograpsus messor (Decapoda: Grapsidae).

Journal of Crustacean Biology 19:361-370.

Bauer RT. 1986. Phylogenetic trends in sperm transfer and storage complexity in decapod

crustaceans. Journal of Crustacean Biology 6: 313-325.

Bauer RT. 2004. Remarkable shrimps adaptations and natural history of the carideans.

Columbia University of Oklahoma press. Normam. Marine Resources Library.

Bauer RT, Martin JW. 1990. Crustacean sexual biology. Columbia University Press. New

York.

Bauer RT, Min LJ. 1993. Spermatophores and plug substance of the marine shrimp

Trachypenaeus similis (Crustacea: Decapoda: Penaeidae): formation in the male

reproductive tract and disposition in the inseminated female. The Biological Bulletin

185: 174-185.

Becker C, Scholtz G. 2017. Phylogenetic implications of sperm storage in Podotremata:

Histology and 3D‐reconstructions of spermathecae and gonopores in female carrier

crabs (Decapoda: Brachyura: Homoloidea). Journal of Morphology 278: 89-105.

Benhalima K, Moriyasu M. 2000. Structure and function of the posterior vas deferens of

the snow crab, Chionoecetes opilio (Brachyura, Majidae). Invertebrate Reproduction

& Development 37: 11-23.

Beninger PG, Elner RW, Foyle TP, Odense P. 1988. Functional anatomy of the male

reproductive system and the female spermatheca in the snow crab Chionoecetes

opilio (O. Fabricius) (Decapoda: Majidae) and a hypothesis for fertilization. Journal

of Crustacean Biology 8:322-332.

Buranelli RC, Mantelatto FL. 2012. Reproductive apparatus of the male giant hermit crab

Petrochirus diogenes (Anomura, Diogenidae): morphology and phylogenetic

implications. Aquatic Biology 16: 241-251.

Buranelli RC, Zara FJ, Mantelatto FL. 2014. Male reproductive system of the red brocade

hermit crab Dardanus insignis (Diogenidae) and its relationship to other family

members. Zoomorphology 133:127-137.

Castilho GG, Ostrensky A, Pie MR, Boeger W A. 2008. Morphology and histology of the

male reproductive system of the mangrove land crab Ucides cordatus (L.)

(Crustacea, Brachyura, Ocypodidae). Acta Zoologica 89:157-161.

Chow S, Dougherty MM, Dougherty WJ, Sandifer PA. 1991. Spermatophore formation in

the white shrimps Penaeus setiferus and P. vannamei. Journal of Crustacean Biology

11:201-216.

Dudenhausen EE, Talbot P. 1983. An ultrastructural comparison of soft and hardened

spermatophores from the crayfish Pacifastacus leniusculus Dana. Canadian Journal

of Zoology 61:182-194.

El-Sherief SS. 1991. Fine Structure of the Sperm and Spermatophores of Portunus

pelagicus (L.) (Decapoda, Brachyura). Crustaceana 61:271-279, 1991.

Erkan M, Tunali Y, Balkis H, Oliveria E. 2009. Morphology of Testis and Vas Deferens in

the Xanthoid Crab, Eriphia verrucosa (ForskÅL, 1775) (Decapoda: Brachyura).

Journal of Crustacean Biology, 29:458-465.


77 Caunesp

Fantucci M.Z, Mantelatto FL. 2011. Male reproductive apparatus and spermatophore

morphology of the hermit crabs Pagurus brevidactylus and P. criniticornis

(Anomura, Paguridae). Journal of Morphology 272:1271-1280.

Garcia TM, Silva JRF. 2006. Testis and vas deferens morphology of the red-clawed

mangrove tree crab (Goniopsis cruentata) (Latreille, 1803). Brazilian Archives of

Biology and Technology 49:339-345.

Guinot D. 1977. — Propositions pour une nouvelle classification des Crustacés Décapodes

Brachyoures. Comptes rendus hebdomadaires des Séances de l’Académie des

Sciences (D) 285: 1049-1052.

Guinot D, Jamieson BGM, Tudge CC. 1997. Ultrastructure and relationships of

spermatozoa of the freshwater crabs Potamon fluviatile and Potamon ibericum

(Crustacea, Brachyura, Potamidae). Journal of Zoology 241:229-244.

Guinot D, Quenette G. 2005. The spermatheca in Podotreme crabs (Crustacea, Decapoda,

Brachyura, Podotremata) and its phylogenetic implication. Zoosystema 27:267-342.

Guinot D, Tavares M. 2003. A new subfamilial arrangement for the Dromiidae de Haan,

1833, with diagnoses and descriptions of new genera and species. Zoosystema,

25:43-129.

Guinot D, Tavares M, Castro P. 2013. Significance of the sexual openings and

supplementary structures on the phylogeny of brachyuran crabs (Crustacea,

Decapoda, Brachyura), with new nomina for higher-ranked podotreme taxa. Zootaxa

3665: 1- 414.

Hartnoll RG. 1964. The freshwater grapsid crabs of Jamaica. Proceedings of the Linnean

Society of London 175:145.

Hartnoll RG. 1969. Mating in the Brachyura. Crustaceana 16:161-181.

Hartnoll RG. 1975. Copulatory structure and function in the Dromiacea, and their bearing

on the evolution of the Brachyura. Publicationes Statione zoologico Napoli 39:657-

676.

Hayer S, Köhnk, S, Boretius S, Brandis D. 2016. Ever more complex: a new type of

organization of reproductive organs in female Dorippe sinica Chen, 1980 (Decapoda:

Brachyura: Dorippidae). Zoology 119: 455-463.

Hinsch GW. 1991. Ultrastructure of the sperm and spermatophores of the anomuran crab

Pleuroncodes planipes. Journal of Crustacean Biology 11:17-22.

Hinsch GW, Walker MH. 1974. The vas deferens of the spider crab, Libinia emarginata.

Journal of Morphology 143: 1-19.

Hobbs HH, Harvey MC, Hobbs HH. 2007. A comparative study of functional morphology

of the male reproductive systems in the Astacidea with emphasis on the freshwater

crayfishes (Crustacea: Decapoda). Washington, D. C.: Smithsonian Institution

Scholarly Press.

Johnson PT. (ed.). 1980. Histology of the Blue Crab, Callinectes sapidus: A Model for the

Decapoda. Praeger, New York.

Junqueira LCU, Junqueira LMMS. 1983. Técnicas Básicas de Citologia e Histologia. 1.

ed. Instituto de Ciências Biomédicas e Faculdade de Medicina, Universidade de São

Paulo. Editora Santos, 123p.

Klaus S, Brandis D. 2011. Evolution of sperm morphology in potamid freshwater crabs

(Crustacea: Brachyura: Potamoidea). Zoological Journal of the Linnean Society

161:53-63.

Klaus S, Schubart CD, Brandis D. 2009. Ultrastructure of Spermatozoa and

Spermatophores of Old World Freshwater Crabs (Brachyura: Potamoidea:

Gecarcinucidae, Potamidae, and Potamonautidae). Journal of Morphology 270:175-

193.


78 Caunesp

Kooda‐Cisco MJ, Talbot P. 1982. A structural analysis of the freshly extruded

spermatophore from the lobster, Homarus americanus. Journal of Morphology

172:193-207.

Kooda‐Cisco M, Talbot P. 1986. Ultrastructure and role of the lobster vas deferens in

spermatophore formation: the proximal segment. Journal of Morphology 188: 91-

103.

Krol RM, Hawkins WE, Overstreet RM 1992. Reproductive components, 295-343. In:

Harrison FW, Humes AG. 1992. (eds.), Microscopic Anatomy of Invertebrates. v. 10:

Decapod Crustacea, Wiley-Liss Inc., New York.

López Greco LS 2013. Functional anatomy of the reproductive system. In: Functional

Morphology and Diversity. Edited by Les Watling and Martin Thiel. Oxford

University Press.

López Greco LS, Vazquez F, Rodríguez EM. 2007. Morphology of the male reproductive

system and spermatophore formation in the freshwater ‘red claw’crayfish Cherax

quadricarinatus (Von Martens, 1898) (Decapoda, Parastacidae). Acta Zoologica

88:223-229.

Mantelatto FL, Scelzo MA, Tudge CC. 2009. Morphological and morphometric appraisal

of the spermatophore of the southern hermit crab Isocheles sawayai Forest and Saint

Laurent, 1968 (Anomura: Diogenidae), with comments on gonopores of both sexes.

Zoologischer Anzeiger 248:1–8.

McLay CL, Becker C. 2015. Reproduction in Brachyura. In: Treatise on Zoology-Anatomy,

Taxonomy, Biology. The Crustacea, Brill, 9:185-243.

McLay CL, López Greco LS. 2011. A hypothesis about the origin of sperm storage in the

Eubrachyura, the effects of seminal receptacle structure on mating strategies and the

evolution of crab diversity: how did a race to be first become a race to be

last?. Zoologischer Anzeiger-A Journal of Comparative Zoology, 4: 378-406.

Mello MSL, Vidal BC. 1980. Práticas de Biologia Celular. São Paulo: Edgar Blucher –

FUNCAMP.

Minagawa M. 1993. Gonopods of the red frog crab Ranina ranina Linnaeus (Decapoda:

Raninidae). Crustacean Research 22:45-54.

Minagawa M, Chiu JR, Kudo M, Takashima F. 1994. Male reproductive biology of the red

frog crab, Ranina ranina, off Hachijojima, Izu Islands, Japan. Marine Biology

118:393-401.

Montanaro J, Gruber D, Leisch N. 2016. Improved ultrastructure of marine invertebrates

using non-toxic buffers. PeerJ 4 : p. e1860.

Nagao J, Munehara H. 2003 Annual cycle of testicular maturation in the helmet crab

Telmessus cheiragonus. Fisheries Science 69:1200-1208.

Nascimento FAD, Zara FJ. 2013. Development of the male reproductive system in

Callinectes ornatus Ordway, 1863 (Brachyura: Portunidae). Nauplius 21:161-177.

Nicolau CF, Nascimento AA, Machado-Santos C, Sales A, Oshiro LMY. 2012. Gonads of

males and females of the mangrove tree crab Aratus pisonii (Grapsidae: Brachyura:

Decapoda): a histological and histochemical view. Acta Zoologica 93:222-230.

Ravi R, Manisseri MK, Sanil NK. 2014. Structure of the male reproductive system of the

blue swimmer crab Portunus pelagicus (Decapoda: Portunidae). Acta Zoologica

95:176-185.

Reynolds ES. 1963. The use of lead citrate at high ph as an electron-opaque stain in

electron microscopy. The Journal of cell biology 17: p. 208.

Ro S, Talbot P, Trujillo JL, Lawrence AL. 1990. Structure and function of the vas deferens

in the shrimp Penaeus setiferus: segments 1-3. Journal of Crustacean Biology

10:455-468.


79 Caunesp

Ryan EP. 1967. Structure and function of the reproductive system of the crab Portunus

sanguinolentus Herbst (Brachyura: Portunidae). I. The male system. Proceedings of

the Symposium on Crustacea at Erkulan, Índia, 1965. Part II. Symposium Series 2:

522 – 544.

Santos CM, Lima GV, Nascimento AA, Sales A, Oshiro LMY. 2009. Histological and

histochemical analysis of the gonadal development of males and females of Armases

rubripes (Rathbun 1897) (Crustacea, Brachyura, Sesarmidae). Brazilian Journal of

Biology 69:161-169.

Scelzo MA Mantelatto FL, Tudge CC. 2004. Spermatophore morphology of the endemic

hermit crab Loxopagurus loxochelis (Anomura, Diogenidae) from the southwestern

Atlantic - Brazil and Argentina. Invertebrate Reproduction and Development 46:1-9.

Simeó CG, Ribes E, Rotllant G. 2009. Internal anatomy and ultrastructure of the male

reproductive system of the spider crab Maja brachydactyla (Decapoda: Brachyura).

Tissue and Cell 41:345-361.

Spears T, Abele LG, Kim W. 1992. The monophyly of brachyuran crabs: a phylogenetic

study based on 18S rRNA. Systematic Biology 41:446-461.

Stewart MJ, Stewart P, Soonklang N, Linthong V, Hanna PG, Duan W, Sobhon P. 2010.

Spermatogenesis in the blue swimming crab, Portunus pelagicus, and evidence for

histones in mature sperm nuclei. Tissue and Cell 42:137-150.

Subramoniam T. 1993. Spermatophores and sperm transfer in marine crustaceans.

Advances in marine biology 29:129-214.

Tavares MS, Secretan S. 1993. — La notion de thelycum et de spermathèque chez les

Crustacés Décapodes. Comptes rendus de l’Académie des Sciences 316: 133-138.

Tiseo GR, Mantelatto FL, Zara FJ. 2014. Is cleistospermy and coenospermy related to

sperm transfer? A comparative study of the male reproductive system of

Pachygrapsus transversus and Pachygrapsus gracilis (Brachyura: Grapsidae).

Journal of Crustacean Biology 34:704-716.

Tiseo GR, Mantelatto FL, Zara FJ. 2017. Ultrastructure of spermatophores and

spermatozoa of intertidal crabs Pachygrapsus transversus, Pachygrapsus gracilis

and Geograpsus lividus (Decapoda: Grapsidae). Zoologischer Anzeiger- A Journal of

Comparative Zoology 269: 166-176.

Tsang LM, Schubart CD, Ahyong ST, Lai JC, Au EY, Chan TY, Ng KLP, Chu KH. 2014.

Evolutionary history of true crabs (Crustacea: Decapoda: Brachyura) and the origin

of freshwater crabs. Molecular biology and evolution 31: 1173-1187.

Tudge CC. 1991. Spermatophore Diversity Within and Among the Hermit Crab Families,

Coenobitidae, Diogenidae, and Paguridae (Paguroidea, Anomura, Decapoda). The

Biological Bulletin 181:238-247.

Vehof J, Scholtz G, Becker C. 2017. Morphology of the female reproductive system of

three dorippid crabs (Crustacea: Decapoda: Brachyura: Dorippidae) and the role of

accessory cuticle structures associated with seminal receptacles. Invertebrate Biology

136: 271-289.

Zara FJ, Caetano FH. 2002. Ultrastructure of the salivary glands of Pachycondyla (=

Neoponera) villosa (Fabricius)(Formicidae: Ponerinae): functional changes during

the last larval instar. Cytologia 67: 267-280.

Zara FJ, Raggi Pereira GR, Sant’Anna BS. 2014. Morphological changes in the seminal

receptacle during ovarian development in the speckled swimming crab Arenaeus

cribrarius. The Biological Bulletin 227: 19-32.

Zara FJ, Toyama MH, Caetano FH, López Greco LS. 2012. Spermatogenesis,

Spermatophore, and Seminal Fluid Production in the adult Blue Crab, Callinectes

danae (Portunidae). Journal of Crustacean Biology 32:249-262.


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List of legends


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Figure 1. Diagram of the male reproductive system of Dromiidae of Brazil. A, testis and

vas deferens of Hypoconcha parasitica and H. arcuata. The pair of testes is located on

both superior sides of the cephalothorax and is continuous with the pair of vasa deferentia,

which extend longitudinally over the hepatopancreas and below the heart, towards the

ventral posterior region of the body (gonopod). Note that the vas deferens does not contain

different anatomical regions, accessory glands or external pouches. B, in M. antillensis, the

pair of testes is also located on both superior sides of the cephalothorax, is continuous with

the pair of vasa deferentia and ends in the posterior region of the body. Observe that the

vas deferens does not exhibit different regions or glands and the posterior part of the vas

deferens is more folded. C, the pair of testes of Dromia erythropus is located on both

superior sides of the cephalothorax and is continuous with the vasa deferentia, ending in

the gonopod. The vas deferens did not show different regions, accessory glands or

pouches. Only the posterior region of the tubule is quite convoluted. T, testes; VD, vas

deferens.

Figure 2. Testis, spermatogenesis and spermiogenesis. A, tubular testis of Hypoconcha

parasitica stained with HE. The seminiferous tubule exhibits germinal, maturation and

evacuation zones with mature spermatozoa. B, the tubular testis of H. arcuata visualized

by toluidine blue. Note the germinal, maturation and evacuation zones. C and D, tubular

testes of Moreiradromia antillensis and Dromia stained with HE. E, germinal center filled

with spermatogonia representing all species. These cells have a large central nucleus

(arrow), several little nucleoli and an acidophilic cytoplasm. F, details of primary

spermatocytes in different stages of the meiotic prophase (arrowheads) of Dromiidae. G,

secondary spermatocytes, with a smaller rounded nucleus and homogeneous chromatin

(arrow) in all species. H, initial spermatids starting spermiogenesis. Observe that these

cells show pro-acrosomal vesicles reactive to PAS (black arrow), producing small vesicles

with homogeneous staining. The nucleus occupies a large part of the volume of these cells

and is basophilic, homogeneous and rounded (white arrow). I, the intermediate spermatids

are marked by the nucleus at the beginning of the formation of the nuclear cup

(arrowheads). Noted in the PAS-positive (white arrow) acrosomal vesicle, a more intensely

marked region (black arrow) indicates the beginning of the formation of the perforatorium

and operculum. J, the final spermatids with a flat nucleus (black arrow) associated with the

acrosomal vesicle (white arrow), both discoid. K, mature spermatozoa with a thin nucleus


82 Caunesp

(black arrow), involving almost the entire extent of the heterogeneous acrosomal vesicle

(white arrow) of H. parasitica, H. arcuata and Moreiradromia antillensis. Note that the

evacuation zone is filled by basophilic secretion, classified as a type I secretion. L, in

Dromia erythropus, the evacuation zone is filled by a type I secretion, which contains

heterogeneous granules near their periphery. These granules diffuse over the basophilic

secretion where the spermatozoa are immersed. EST, early spermatid; EZ, evacuation

zone; GZ, germinal zone; LST, late spermatid; MST, middle spermatid; MT, maturation

zone; SI, type I secretion; SPG, spermatogonia; SPC1, spermatocytes I; SPC2,

spermatocytes II; SZ, spermatozoa.

Figure 3. Hypoconcha parasitica vas deferens . A, in a longitudinal section, the vas

deferens with HE is a single tubule, continuous and without differentiation. The

spermatozoa are immersed in type I secretion and compacted by type II secretion with the

presence of a fibrous material (**), forming a large spermatic cord, producing a unique

extremely elongated kind of coenosoermic “spermatophore”. Note the simple epithelium

(arrowheads), supported on a thin layer of connective tissue, surrounded by marked

musculature. B, general aspect of the vas deferens ultrastructure, showing spermatozoa in a

homogeneous type 1 secretion, which is surrounded by a finely granular type II secretion,

both with different electron densities. C, the cross-section stained with HE confirms the

tubular morphology of the vas deferens. Note that the spermatozoa in the lumen of the

tubule are surrounded by type II secretion, which is thin in the regions near the testis and

thicker along the vas deferens. The rectangular area is shown in panels D, E, F and G. D,

vas deferens submitted to HE. The basophilic spermatozoa are immersed in acidophilic

type I secretion. The type II secretion is basophilic, and the fibrous material is acidophilic

(**). E, Vas deferens to ponceau xylidine, with positive reactions in type I secretion. The

type II secretion is stained strongly for proteins. Note that the fibrous material strongly

protein (**). F, type I secretion and connective tissue (arrow) reactive to neutral

polysaccharides. Note that type II secretion is strongly reactive to PAS, such as the fibrous

material (**). G, when stained with Alcian Blue, the type I and II secretions were positive

and weakly reactive to acidic polysaccharides, respectively. Note the convolute vas

deferens within the type II secretion. EP, epithelium; M, muscular layer; SI, type I

secretion; SII, type II secretion; SZ, spermatozoa.


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Figure 4. Hypoconcha arcuata vas deferens. A, longitudinal section of the vas deferens

by HE, showing that the spermatozoa are immersed in type I secretion and are compacted

by type II secretion, forming a large spermatic cord, producing a unique extremely

elongated coenosoermic spermatophore. B, Ultrastructure of the vas deferens. Note that the

spermatozoa are immersed in an electron-dense and strongly granular type I secretion,

where they are compacted by a homogeneous type II secretion. C, The cross-section shows

that the vas deferens has no histological variations or different cell types from the anterior,

middle and posterior regions stained with HE. Note the simple epithelium on a thin layer of

connective tissue surrounded by strong musculature, displaying multiple layers. The

rectangular area is shown in panels D, E, F and G. D, the longitudinal section of the vas

deferens by HE shows that the type I secretion is weakly basophilic, and the type II

secretion is acidophilic. E, the type I and II secretions are slightly reactive to ponceau

xylidine and to F, PAS. G, type I and II secretions are negative for acidic polysaccharides

stained with Alcian Blue. Note that the spermatozoa are only reactive when stained. EP,

epithelium; M, muscular layer; SI, type I secretion; SII, type II secretion; SZ, spermatozoa.

Figure 5. Moreiradromia antillensis vas deferens. A, longitudinal section of the vas

deferens, showing the extremely elongated kind of coenosoermic spermatophore stained

with HE. The spermatozoa are immersed in a type I secretion, which is surrounded by the

type II secretion, composed of heterogeneous granules in a finely granular matrix

(arrowhead). Note that the vas deferens is formed by a simple epithelium, on a thin

connective layer, covered by musculature. B, the ultrastructure of the vas deferens shows

the spermatozoa in an electron-dense and homogeneous SI secretion, compacted by a type

II secretion that is less electron-dense than a type I secretion, with the presence of

homogeneously electron-dense granules. C, cross-section of the vas deferens, confirming

the continuous and tubular morphology of the elongated spermatophore. The rectangular

area is shown in panels D, E, F and G. D, spermatozoa immersed in basophilic type I

secretion, which is compacted by a granular and heterogeneous type II secretion. Observe

that the type II secretion matrix is basophilic (arrowhead). E, vas deferens submitted to the

ponceau xylidine. The type I secretion and type II secretion matrix are slightly reactive to

proteins (arrowhead). Note the heterogeneous granules of the type II secretion is strongly

reactive to proteins. F, the type I secretion and the secretion matrix II (arrowhead) are

weakly reactive to neutral polysaccharides with PAS. G, when submitted to Alcian Blue,


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the type I secretion is slightly reactive, and the type II secretion and its matrix (arrowhead)

are negative for the stain. EP, epithelium; M, muscular layer; SI, type I secretion; SII, type

II secretion; SZ, spermatozoa.

Figure 6. Dromia erythropus vas deferens. A, the lumen of the vas deferens with HE

shows that the anterior region spermatozoa are immersed in type I secretion and

surrounded by a type II secretion, which is covered by types III and IV secretions. This

arrangement of secretions forms an elongated coenosoermic spermatophore and is the most

complex type of structuring among all the species in this study. B, under transmission

electron microscopy, the spermatic cord is formed by a type I secretion (electron-dense and

homogeneous), and the spermatozoa are compacted by a type II secretion, which shows

electron-dense granules. The type III secretion is electron-dense, with discrete electron-

lucid granules surrounding the type II secretion, and is covered by the type IV secretion,

which exhibits a portion that is electron-dense and another that is less electron-dense. C,

the cross-section of the vas deferens in HE shows the unique and continuous structure of

the elongated spermatophore. Note that the spermatozoa in the vas deferens lumen are

immersed in type I secretion. The other type II, III and IV secretions are added externally

to the lumen of the vas and are thicker near the posterior region. The rectangular area is

shown in panels D, E, F and G. D, vas deferens by HE, showing that the spermatozoa are

immersed in the weakly basophilic type I secretion. Note the granular type II secretion,

being the limit of these granules marked by acidophilic secretion. Observe that the type III

secretion is slightly basophilic, and the type IV secretion is composed of two layers: one

external to the basophilic type III secretion (white arrow) and another acidophilic next to

the epithelium (black arrow). E, submitted to ponceau xylidine the type I, II, III secretions

are weakly, strongly and negatively reactive to proteins, respectively. The outer layer of

the type III secretion is slightly reactive to proteins (white arrow), and the other layer next

to the epithelium is positive for proteins (black arrow). F, vas deferens by PAS. Note that

the type I secretion is weakly reactive to the stain, and the type II shows a negative

reaction. The type III secretion is slightly positive, and the first layer of type IV secretion

(the one external to the type III secretion) is strongly positive (white arrow). The second

layer (next the epithelium) is positive to the stain (black arrow). G, type I and type II

secretions are negative for acidic polysaccharides when stained with Alcian Blue. Note that

the type III secretions are weakly reactive to acidic polysaccharides. The first and second


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layers of secretion IV are slightly reactive (white arrow) and negative (black arrow),

respectively, when stained by this technique. EP, epithelium; SI, type I secretion; SII, type

II secretion; SIII, type III secretion; SIV, type IV secretion; SZ, spermatozoa.

Figure 7. Ultrastructure of Dromiidae vas deferens. A, the Dromiidae vasa deferentia

studied show the same cell ultrastructure along the tube; thus, we use the cells of H.

parasitica as a model for the description. The vas deferens wall was composed of an

external connective layer, a middle muscular layer and an internal epithelium, and the

lumen is filled by a less electron-dense secretion where the spermatozoa are distributed and

other secretions, which show different electron-densities depending on the species. Note

that the thin connective tissue layer contained fibroblast-like cells with oval or flat nuclei.

The rectangular areas (1 and 2) are shown in panel B and C, respectively. The rectangular

(3) shows the panels D, E and F, and the rectangular (4) is shown in panels G and H. B, the

cytoplasm contains polyribosomes, rough endoplasmic reticulum (RER) and some

mitochondria. Observe that one layer is composed of striated muscular fibers, showing at

least one longitudinal fiber and other oblique fibers. C, epithelial cells show a thick basal

lamina, which is filled with components of different electron-densities, and the base of the

epithelial cell depicts many deep basal plasma membrane folds and many mitochondria

and polyribosomes. D, the cytoplasm is filled with a large amount of RER, and the nucleus

is positioned at the middle of the cell. Note that the nucleus is flattened, showing more

condensed chromatin associated with the nuclear envelope (arrows). E, RER composed of

many parallel cisternae, and among them, there are a lot of mitochondria and well-

developed Golgi complexes. F, the well-developed Golgi complexes are present in the

epithelium cytoplasm. Note that this organelle produces vesicles that are released to the

lumen by the apical region of the cell (arrows). G, the Golgi complexes produce at least

two types of vesicles, one electron lucent (white arrow) and another filled with granular

and electron-dense material (black arrow). H, the epithelium shows small microvilli, which

are regularly distributed on the surface. Note the vesicles produced by the Golgi complexes

being released to the lumen by exocytosis at the apical region. BL, basal lamina; CG, Golgi

complex; CY, cytoplasm; EP, epithelium; L, lumen; M, musculature; N, nuclei; MT,

mitochondria; MV, microvilli; RER, rough endoplasmic reticulum; S, secretion; SZ,

spermatozoa; TC, connective tissue.


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Figure 8. Scanning electron microscopy of the penial tube and two gonopod (G2) in

Dromiidae. Mobile penial tube of A, H. parasitica and B, H. arcuata, showing the long

plumose setae mainly located in the basal portion (black arrows) and on one of the side

margins (white arrow). The penial tube is longer, flat dorso-ventrally, and slightly curved

at the apex in C, M. antillensis and D, D. erythropus. In this latter species, the basal setae

surround the penial tube, and a line of setae occur along the central 2/3 of the margin and

exhibit pappose setae only on the lateral margin until the apex (arrow). E, the penial tube

apices of H. parasitica show a very thin cuticle, noticeable by the wrinkles caused by

dehydration, which form a mobile operculum. F, the penial tube apex of H. parasitica

showing the operculum. G, Moreiradromia antillensis penial tube apex forming the

operculum. H, the penial tube apex of D. erythropus showing the operculum. I, the

fractured G2 of M. antillensis representing all species studied here, showing the secretion

that forms a smooth surface around the spermatozoa. J, details of G2 fractured apex of M.

antillensis. Note the thinner secretion due to the dehydration process (white arrow) and

spermatic cord portion (black arrow). G2, two gonopod; PT, penial tube.

Figure 9. Hypoconcha parasitica and Moreiradromia antillensis seminal fluid under DIC

microscope. The seminal fluid squeezed from the vas deferens maintains the structure,

forming a sperm cord showing spermatozoa immersed in the type I secretion surrounded

by the type II secretion in both A, Hypoconchinae and B, Dromiinae. SI, type I secretion;

SII, type II secretion; SZ, spermatozoa; VD, vas deferens.

Figure 10. “Sperm plug” and female first pleopod of H. parasitica and M. antillensis.

A, overview of H. parasitica sternum showing the location of the pair of first pleopod, the

spermathecal aperture and the first, second and third pereopods. Note a material similar to

a sperm plug (**) on the spermathecae aperture, with traces of spermatozoa and the

gonopores on the third coxae. B, details of the first pleopods of H. parasitica, in which

they are almost perpendicular to the body axis but not reaching the spermathecal apertures,

and their lateral edge apices are fringed with setae. C, the material, similar to a “sperm

plug” (**), included traces of spermatozoa in H. parasitica. D, details of the spermatozoa


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found in the material on the spermathecal aperture of H. parasitica. Note the spermatozoon

with a centrally perforated operculum. E, the first pleopod in M. antillensis is present in the

1/3 portion of the spermathecae, not reaching the spermathecal aperture. Note the first,

second and third pereopod in the female sternum. F, the first pleopod of M. antillensis,

showing the edge apex also fringed with setae. G, ovigerous female M. antillensis with

material similar to a sperm plug (**) on the spermathecal aperture. H, the material found in

the aperture of spermathecae of M. antillensis showing traces of spermatozoa (**). I,

details of the spermatozoa found in the material on the spermathecal aperture of M.

antillensis, fixed in 100% alcohol. I, spermatozoa fixed in 4% paraformaldehyde prepared

with seawater and 0.2 M sodium phosphate buffer. AP, spermathecal aperture; GO,

gonopore; N, nucleus; O, operculum; Pl1, first pleopod; P1, first pereopod; P2, second

pereopod; P3, third pereopod; P4, fourth pereopod; P5, fifth pereopod; SZ, spermatozoa.


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List of figures and table


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Fig. 1


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Fig. 2


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Fig. 3


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Fig. 4


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Fig. 5


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Fig. 6


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Fig. 7


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Fig. 8


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Fig. 9


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Fig 10.


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Table 1: Histochemistry of spermatozoa and secretions from the vas deferens in Hypoconcha parasitica, H. arcuata,

Moreiradromia antillensis and Dromia erythropus. +++ = strongly positive; ++ = positive; + = weakly positive; - = negative.

Ponceau xylidine PAS (neutral Alcian blue (acidic

(total proteins) polysaccharides) polysaccharides)

Hypoconcha parasitica Spermatozoa ++ ++ +++

Type I secretion + ++ ++

Type II secretion +++ +++ +

Fibrous material +++ +++ −

Hypoconcha arcuata Spermatozoa +++ +++ ++

Type I secretion ++ ++ +

Type II secretion +++ + −

Moreiradromia antillensis Spermatozoa +++ +++ +++

Type I secretion ++ ++ ++

Type II secretion (granular) +++ − −

SII matrix ++ ++ +

Dromia erythropus Spermatozoa +++ +++ +

Type I secretion + ++ −

Type II secretion (granular) +++ − −

Type III secretion − ++ ++

Type IV secretion (external to SIII) + +++ +++

Type IV secretion (next to

epithelium) ++ ++ −


Capítulo redigido de acordo com as normas do periódico Acta Zoologica Caunesp

Capítulo III

Comportamento de cópula e armazenamento espermático no

caranguejo Hypoconcha parasitica (Podotremata: Dromiidae)

Maria Alice Garcia Bento & Fernando José Zara


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Comportamento de cópula e armazenamento espermático no caranguejo Hypoconcha

parasitica (Podotremata: Dromiidae)

Maria Alice Garcia Bento¹ & Fernando José Zara¹

¹ Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), FCAV, Departamento de

Biologia Aplicada, Laboratório de Morfologia de Invertebrados (IML), Via de Acesso Prof. Paulo

Donato Castellane, s/n, Jaboticabal, 14884-900, São Paulo, Brazil. E-mails:

[email protected]; [email protected].

Condensed title: Comportamento de copula e armazenamento espermático em Dromiidae




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RESUMO

Neste trabalho descrevemos o comportamento de cópula de Hypoconcha parasitica, em

condições laboratoriais, e adicionalmente, analisamos a morfologia da espermateca e o

padrão de armazenamento dos espermatozoides e fluido seminal. Os casais foram mantidos

em aquários e os comportamentos de acasalamento foram filmados e quantificados. As

espermatecas foram processadas seguindo as rotinas para microscopia eletrônica de

varredura, histologia e histoquímica, com prévio tratamento em EDTA. O comportamento

de corte e guarda copulatória estão ausentes em H. parasitica. A transferência espermática

ocorreu enquanto os casais encontravam-se em “postura bivalve”. Três cópulas foram

registradas, com machos sempre em intermuda, enquanto uma fêmea copulou em muda e

duas em intermuda. A espermateca é uma invaginação dos seguimentos torácicos esternos

7/8, recoberta exclusivamente por cutícula, seguindo o padrão de Podotremata.

Externamente a parede da espermateca notam-se fibras musculares associadas à cutícula,

principalmente nas regiões mais distais, oposta a abertura. A organização da espermateca

indica que o processo de liberação dos espermatozoides para a fertilização ocorre por meio

de ação muscular exercida na parede da câmara. Assim, a distribuição da musculatura em

Hypoconchinae é diferente do descrito para o Homolidae Paromola cuvieri, a qual é

concentrada na abertura da espermateca. Como em Homolidae, o pleopodo 1 parece estar

envolvido na movimentação de espermatozoides e ovócitos no momento da fertilização em

H. parasitica. Assim, a morfologia da espermateca e estruturas associadas trazem novas

informações sobre os mecanismos envolvidos na reprodução de caranguejos primitivos e

como ocorreu a modificação desta estrutura armazenadora entre os Podotremata.

Correspondence: Maria Alice Garcia Bento, UNESP, Faculdade de Ciências Agrárias e

Veterinárias- Campus de Jaboticabal, Departamento de Biologia Aplicada, Laboratório de

Morfologia de Invertebrados (IML), Via de Acesso Prof. Paulo Donato Castellane, s/n,

Jaboticabal, 14884-900, São Paulo- SP, Brazil. E-mail: [email protected]



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INTRODUÇÃO

O comportamento de acasalamento tem sido estudado em diversos crustáceos

Decapoda (Hartnoll, 1969; Waddy & Aiken, 1990; Diesel, 1991; Bauer, 1996, Pinheiro &

Fransozo, 1999). Entretanto, este assunto é pouco conhecido para os caranguejos

primitivos pertencentes à seção Podotremata Guinot, 1977. Esta seção agrupa

aproximadamente 89 gêneros em que sistema reprodutor feminino e masculino abrem-se

por meio de orifícios coxais (gonóporos), pertencentes aos terceiros e quintos pereópodos,

respectivamente (Guinot & Tavares, 2001; Guinot & Quenette, 2005; Ng et al., 2008;

Guinot et al., 2013). Adicionalmente, as fêmeas portam um par de estruturas que são

responsáveis pelo armazenamento dos espermatozoides até a fertilização, denominadas

espermatecas (Tavares & Secretan, 1993; Guinot & Quenette, 2005; Guinot et al., 2013).

A cópula foi somente estudada no Dromiidae Dromia personata (Linnaeus, 1758) e

no Raninidae Ranina ranina (Linnaeus, 1758) dentre os Podotremata (Hartnoll, 1975;

Skinner & Hill, 1987; Guinot et al., 2013). Em D. personata o acasalamento pode ocorrer

com fêmeas em muda e em intermuda, enquanto que em R. ranina, a cópula ocorre com

fêmeas em intermuda, sendo que ambas espécies não exibem comportamentos de corte e

guarda (Hartnoll, 1975; Skinner & Hill, 1987). Para as espécies de Eubrachyura, dois

principais padrões de acasalamento foram estabelecidos. O primeiro padrão é aquele em

que o acasalamento ocorre rapidamente após a muda da fêmea, quando estas encontram-se

sob a condição de exoesqueleto mole (Hartnoll, 1969; Christy, 1987). Neste caso, os

comportamentos de corte e guarda prolongada são bastante comuns (Hartnoll, 1969;

Christy, 1987; Jivoff & Hines, 1998; Mclay & López Greco, 2011). No segundo padrão, o

acasalamento ocorre quando as fêmeas encontram-se em intermuda (exoesqueleto rígido) e

o comportamento de corte (quando presente) é breve e a guarda prolongada normalmente

está ausente (Hartnoll, 1969; Christy, 1987).


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A espermateca tem origem exclusivamente ectodérmica, sendo constituída por

invaginações dos seguimentos torácicos esternos adjacentes 7/8, nos quais formam uma

depressão no esterno, cuja abertura é alongada em forma de fenda, sem apresentar ligação

interna com o oviduto (Guinot & Tavares, 2001; Guinot & Tavares, 2003; Guinot &

Quenette, 2005; Guinot et al., 2013). Desta maneira, os ovócitos dos Podotremata são

ovulados via gonóporos coxais e os espermatozoides são liberados da espermateca para

que a fertilização externa ocorra (Hartnoll, 1968, 1969; Guinot & Quenette, 2005; Guinot

et al., 2013; Mclay & Becker, 2015). Este padrão encontrado em Podotremata é diferente

ao observado nos caranguejos Eubrachyura, nos quais as fêmeas portam gonóporos nos

esternos do sexto segmento torácico e são ligados internamente aos receptáculos seminais

de origem ecto- mesodérmica. O receptáculo seminal recebe o oviduto, por meio de uma

conexão dorsal ou ventral, tendo papel importante na competição espermática (Diesel,

1989, 1991, McLay & López-Greco, 2011; Antunes et al., 2016). Assim, a fertilização em

Eubrachyura ocorre internamente ao corpo destes, podendo ocorrer nos receptáculos

seminais ou em outras estruturas internas associadas (Hartnoll, 1968, 1969; Guinot et al.,

2013; Mclay & Becker, 2015; Hayer et al., 2016).

Apenas dois trabalhos descrevem a histologia da espermateca em Podotremata, sendo

um deles apresentado de maneira esquemática (Hartnoll, 1975; Becker & Sholtz, 2017). O

processo de liberação dos espermatozoides da espermateca para a fertilização externa

parece ocorrer meio de ação muscular, e foi proposto somente para a Homolidae (Becker

& Sholtz, 2017). Assim, o mecanismo de liberação dos espermatozoides da espermateca

em Dromiidae ainda é desconhecido e, com a lacuna de conhecimento da morfologia

funcional desta estrutura, não é possível traçar hipóteses sobre como ocorre à fertilização

em Podotremata (Becker & Sholtz, 2017). Desta maneira, no presente trabalho realizamos

a descrição detalhada do comportamento de cópula e da morfologia da espermateca de


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Hypoconcha parasitica (Linnaeus, 1763), a fim de elucidar os mecanismos de fertilização

nesta espécie de Dromiidae e verificar se os comportamentos exibidos durante a

transferência espermática e a maneira como os espermatozoides são armazenados no

interior da espermateca são semelhantes ao padrão comumente encontrado para

caranguejos Brachyura.

MATERIAL E MÉTODOS

Animais

Machos e fêmeas maduras de Hypoconcha parasitica de 19,0 a 22,0 mm de largura

da carapaça (LC) foram coletados no município de Ubatuba, São Paulo, Brasil

(25°07´385´´S/47°52´508´´W) no período de agosto de 2016 a novembro de 2017, por

meio de arrasto (20 min.), utilizando-se barco de pesca camaroneira, com redes do tipo

“double rig”, a uma profundidade de 10 a 18 metros. Posteriormente, os animais foram

transportados vivos em caixas de isopor com a devida aeração para o Laboratório de

Morfologia de Invertebrados do Departamento de Biologia Aplicada à Agropecuária,

FCAV, UNESP- Jaboticabal, onde foram mantidos em aquários para devida manutenção.

Comportamento reprodutivo

As análises do comportamento reprodutivo de H. parasitica foram realizadas em

condições laboratoriais. Para tal, os casais foram mantidos em aquários (45x30x20cm) com

um casal cada, em água do mar aerada (salinidade de 33 a 35 Psu e temperatura média de

25ºC). Para a separação dos casais nos aquários, foram utilizadas telas plásticas removíveis

para que o contato químico e visual fossem mantidos. Para identificar o período de

preferência de cópula, a tela plástica foi removida cinco vezes no período entre 09:00h e


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18:00h e cinco vezes entre 19:00h e 05:00h da manhã. Para observações em período

noturno, os aquários foram iluminados por luz infravermelha (Pinheiro e Fransozo, 1999).

Além disso, os períodos de contato entre os animais após a remoção da tela removível,

foram adicionalmente registrados por meio de câmera digital (GoPro Hero 5 e Sony DCR

HC40). Sedimentos arenosos e conchas de bivalves foram utilizados como substrato, para

mimetizar o ambiente natural. Todas os animais utilizados encontravam-se em intermuda,

exceto em um evento em que a fêmea sofreu muda durante a experimentação, estando

assim, com o exoesqueleto não calcificado (carapaça mole). As filmagens foram

armazenadas em cartão de memória e posteriormente analisadas em computador para

averiguação do repertório comportamental reprodutivo de H. parasitica. Ao término da

cópula, foi realizada a checagem da região ventral das fêmeas para verificação do aspecto

da espermateca quanto a presença de plug espermático. As fêmeas que copularam nos

aquários foram checadas a cada duas horas nas primeiras 24h e após este período, a

checagem foi realizada a cada dia, para verificar o momento do evento de ovulação.

Análise da espermateca e ovários

Para as análises histológicas e ultraestruturais, seis fêmeas tiveram uma de suas

espermatecas fixadas em solução Karnovsky (glutaraldeído 2,5% e parafolmaldeído 2%

em tampão cacodilato de sódio 0.1M, pH 7.4 (Karnovski, 1965), com a adição de sacarose

5% (Ro et al., 1990) durante três a quatro dias a 4ºC (pH 7.4). As outras espermatecas,

bem como os ovários foram fixadas em paraformaldeído 4% em tampão fosfato de sódio

0.1M (pH7.4) pelo mesmo período. Após a fixação em Karnovsky, as amostras foram

lavadas três vezes em tampão cacodilato de sódio 0.1M (pH 7.4), com duração de 10

minutos cada e pós - fixadas em tetróxido de ósmio 1% no mesmo tampão por 30 minutos.

Posteriormente, os materiais foram desidratados em séries crescentes de álcool (70 a


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100%) por 15 minutos cada. Quando em álcool 100%, as espermatecas foram envolvidas

em Parafilm (Bemis) e congeladas rapidamente em nitrogênio líquido. Após o

congelamento, com uma lâmina previamente congelada, o material foi fraturado, sendo

posteriormente descongelado no álcool 100% (Hayat, 1978). Em seguida as amostras

foram submetidas a completa secagem em ponto crítico CPD 030 (Balzer Union), com

CO2 líquido. Posteriormente, foram fixadas em “stubs” de alumínio e levadas ao

metalizador (SC 070 - Balzer Union) para serem vaporizadas com ouro (camada de 10

milímetros). Ao final, todas as amostras foram analisadas e fotografadas em microscópio

eletrônico de varredura Zeeis EVO 10, com intensidade do feixe de elétrons variando de 10

- 20KV.

Para as análises histológicas e histoquímicas, as espermatecas das seis fêmeas

fixadas em paraformaldeído 4%, sendo uma após a cópula e outras duas após a ovulação

foram descalcificadas em ácido etilenodiaminotetracético (EDTA) 10% por 48 a 72 horas e

desidratadas em séries crescentes de etanol de 70 a 90%. Após a desidratação, as amostras

foram embebidas e incluídas em historesina glicol-metacrilato Leica®. Os cortes seriados

foram obtidos em micrótomo rotativo (5 a 7µm). Para descrição histológica, o material foi

corado com Hematoxilina & Eosina (Junqueira & Junqueira, 1983) e para a histoquímica,

as lâminas foram submetidas às técnicas de Xylidine Ponceau (Mello & Vidal 1980) para

evidenciar proteínas totais e PAS e Azul de Alcian 2.5% (Junqueira & Junqueira, 1983)

para polissacarídeos neutros e ácidos, respectivamente.

RESULTADOS

Comportamento reprodutivo

Os casais submetidos às análises de cópula apresentavam LC de 19,8 a 25 mm para

os machos, enquanto as fêmeas apresentaram LC variando 19 a 21, 5 mm. Das cinco


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seções de tentativa de pareamento, somente em três a cópula foi efetivada, sendo este

evento sempre durante o dia, duas no período da manhã e uma no final da tarde. Nenhuma

cópula foi observada nos experimentos durante o período da noite. Das três cópulas

realizadas em laboratório, duas aconteceram com fêmeas em estágio de intermuda e uma

em estágio de muda. O LC dos casais foram selecionados de maneira aleatória, sendo que

os casais que efetivamente copularam apresentaram a seguinte proporção macho e fêmea

de LC: 20mm: 21,10 mm; 21, 4 mm: 19,8mm; 25,0 mm: 21, 5mm.

Logo após a remoção da tela removível que separava os casais, os animais

continuaram caminhando normalmente sobre o substrato (Fig.1A). Nenhum tipo de corte

elaborada foi observado até o momento da cópula. Em todas as seções de tentativa de

pareamento, os machos ao encontrar a fêmea, tocavam o seu corpo e iniciaram o

comportamento de escalada sobre a concha da fêmea. Para os três casais que efetivamente

copularam, o macho encontra a fêmea e toca a sua concha com um dos quelípodos e

posteriormente, dirige o outro quelípodo até a concha. Imediatamente, o macho inicia a

escalada sobre a concha da fêmea, a qual se mantém estática ou com movimentos limitados

(Fig. 1B, C). Os pereópodos dois e três do macho estão sempre em contato com a concha

e/ou substrato. Posteriormente, os machos passam a se posicionar em cima da concha da

fêmea. O macho começa a tocar a região ventral da concha onde encontra-se a fêmea.

Posteriormente, o macho apoia a sua concha em posição lateral ao subtrato e com ação dos

quelípodos e pereópodos, ao mesmo tempo que começa a levantar a fêmea, empurrando o

corpo e a concha da mesma (Fig. 1D). Ao levantar completamente a fêmea, o casal atinge a

“postura bivalve”, momento no qual o macho com o os pereópodos dois e três, abre o

abdômen da fêmea, posicionando o seu abdômen sobre o da mesma (Fig. 1E). O tempo

entre os primeiros comportamentos e a postura bivalve foi de 4 a 5 minutos. Antes de

alcançar a postura bivalve, algumas fêmeas não permitiram sua imobilização pelos machos


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e iniciaram um comportamento de fuga, ou seja, comportamento de evitação da cópula, o

que ocorreu em duas seções comportamentais. Quando o macho atinge a posição bivalve,

este insere os segundos gonópodos na abertura da espermateca da fêmea, iniciando a

transferência espermática (Fig. 1E, F). A transferência espermática é marcada pelo

movimento contínuo do abdômen do macho, em vai e vem. Dois tipos de postura bivalve

foram observados, um no qual o macho e fêmea com a região anterior voltada para o

substrato, enquanto que a região posterior do corpo permanece elevada (Fig. 1E), o qual foi

observada uma única vez. A segunda postura bivalve foi observada duas vezes e nesta, o

macho posiciona-se sobre o corpo da fêmea, a qual permanece em decúbito dorsal, com a

concha apoiada sobre o substrato (Fig. 1F). A duração média de cópula foi de 173,3 ± 70

minutos. O término da transferência espermática é marcado pelo movimento dos

quelípodos dos machos em direção à parte anterior do seu corpo, a fim de abandonar a

postura bivalve, ao mesmo tempo que a fêmea move seus pereópodos em direção ao

substrato (Fig. 1G). Ao alcançarem o substrato, os casais se separam e voltam a se mover

livremente no aquário (Fig. 1H). Os machos, sempre em intermuda, copularam com duas

fêmeas em intermuda, as quais tiveram a duração de cópula em 100 e 180 minutos. Em um

só evento de cópula, com a fêmea em muda, a cópula ocorreu durante o período de 240

minutos.

Espermateca e estruturas associadas

A espermateca é um órgão par, localizada na superfície ventral do cefalotórax, mais

especificamente presente na região inclinada da parte posterior do esterno e é constituída

por suturas torácicas derivadas de invaginações dos integumentos externos 7/8 (Fig. 2A,

B). As porções mais posteriores das suturas têm início no mesmo nível da coxa dos

pereópodos quatro (P4), e estendem-se próximo ao nível do par de gonóporos, os quais são

estruturas independentes da espermateca, presentes nas coxas dos terceiros pereópodos


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(P3) (Fig. 2A, B). Acima da extremidade posterior da espermateca existe uma região curta

e estreita, denominada por tubo espermatecal ou câmara de armazenamento, onde,

internamente, o fluido seminal permanece armazenado até o processo de fertilização (Fig.

2A, B). O tubo espermatecal é contínuo e termina na abertura da espermateca, a qual está

localizada na região anterior do esterno torácico, próximo ao P3 (Fig. 2A, B). Em fêmeas

não ovígeras, a abertura da espermateca é oval e pequena (Fig. 2A). Enquanto que em

fêmeas logo após a ovulação (ovígeras), esta região apresenta preenchida por material

seminal (Fig. 2B). Ao microscópio eletrônico de varredura nota-se que a abertura da

espermateca encontra-se no mesmo nível do gonóporo operculado na altura da coxa do

terceiro pereópodo (Fig. 3A). O gonóporo tem forma arredondada e é recoberto por

opérculo membranoso (Fig. 3B). Ao redor do gonóporo nota-se inúmeras cerdas coxais do

tipo plumosas (Fig. 3A, B). A abertura da espermateca em fêmeas logo após a ovulação

apresenta ejaculado no seu interior, onde observam-se claramente os com espermatozoides

(Fig. 3C, D). Em cortes longitudinais ao eixo ântero-posterior da espermateca, nas fêmeas

que acasalaram sob condições laboratoriais, observa-se a presença de fluido seminal com

espermatozoides no seu interior (Fig. 3E, F). Uma das faces da espermateca apresenta

cutícula mais delgada em relação a outra e, nota-se a inserção das fibras musculares

principalmente nas regiões mais distais, ou seja, no lado oposto da abertura da

espermateca, sobre a face cuticular mais espessa (Fig. 3E, 4A, B).

Por meio da histologia, a abertura da espermateca mostra-se estreita e curta, com

curvatura, sem a presença de botões ou estruturas membranosas cuticulares que a separam

da região onde os espermatozoides encontram-se armazenados (Fig. 4A, B). A espermateca

da fêmea dissecada logo após a cópula, bem como fêmeas sacrificadas logo após a

ovulação, mostraram a presença de secreção seminal com espermatozoides junto da

abertura da espermateca. A espermateca mostra espaço interno amplo, a qual se torna cada


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vez mais estreito na direção posterior (Fig. 4A, B). A musculatura corpórea está

diretamente ancorada na face de cutícula mais espessa da espermateca e as células

epidérmicas mostram aspecto pavimentoso e em certos pontos descontínua, o que devido

ao poder de resolução do microscópio de luz assemelha-se a um contato direto da

musculatura na cutícula (Fig. 4C). Por sua vez, a epiderme associada a porção cuticular

mais delgada da espermateca apresenta epitélio colunar simples, com núcleos basais e

citoplasma acidófilo (Fig. 4D). No lúmen da espermateca notam-se dois tipos de secreção,

a secreção do tipo um (SI), onde estão imersos os espermatozoides e a secreção do tipo

dois (SII), sem a presença de espermatozoides. A SI não sofreu alteração histoquímica em

relação às fêmeas recém-copuladas (Fig. 5A - D) e as fêmeas sacrificadas após a ovulação

(Fig. 5E - H), sendo esta uma secreção glicoproteica, com pequena reatividade para

polissacarídeos ácidos (Fig. 5E- H). A secreção tipo II na fêmea recém-copulada é

acidófila, intensamente reativa para proteínas e positiva para polissacarídeos neutros, sendo

negativa para polissacarídeos ácidos (Fig. 5A - D). Porém, nas fêmeas após a ovulação, a

SII apresentou modificação histoquímica, onde esta passou a ser basófila, menos

intensamente corada para proteínas e com intensa reação para polissacarídeos neutros (Fig.

5E - H). Não houve alteração da reação para polissacarídeos ácidos (Fig. 5H).

As fêmeas que copularam no estado de intermuda possuem ovócitos em estágio

avançado de desenvolvimento, com a presença de grande quantidade de grânulos de vitelo

em seu interior (6A, B). A única fêmea que copulou no estágio de muda apresentou

ovócitos rudimentares, caracterizados por citoplasma fortemente basófilo, com várias

dilatações menos basófilas no seu interior (Fig. 6C). A análise dos pleópodos das fêmeas

mostrou que o primeiro par (PL1) não carrega ovos em fêmeas ovígeras, permanecendo

livres na câmara de incubação (Fig. 6D). Os PL1 são curtos, com a região distal cônica e

circundados por inúmeras cerdas plumosas, particularmente abundantes na região apical


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(Fig. 6E). Estes pleópodos são unirremes e com o abdômen fechado se aproximam da

abertura da espermateca (Fig. 6F, G).

DISCUSSÃO

Em Podotremata, o comportamento de cópula foi pouco estudado, sendo as

descrições restritas aos representantes de Dromiidae D. personata e ao Raninidae R. ranina

(Skinner & Hill, 1987; Guinot et al., 2013). Em Hypoconcha parasitica não foi verificado

nenhum tipo de comportamento de corte e todos os machos ao encontrar a fêmea iniciaram

a tentativa de cópula, não havendo evidência de sinais visuais, como observado em outros

Eubrachyura (Ryan, 1967; Hartnoll, 1969, Dunham, 1978; Kennedy & Cronin, 2007).

Assim, H. parasitica seguiu o padrão conhecido para os outros Podotremata (Hartnoll,

1975; Skinner & Hill, 1987). Em H. parasitica uma característica interessante da cópula

foi o posicionamento do macho segurando a fêmea levando a junção das conchas sobre o

corpo ou postura bivalve, que em dois casos, o término da cópula ocorreu com o macho

sobre a fêmea. Nos outros Podotremata descritos na literatura, o macho posiciona a fêmea

sobre o seu corpo, estando este em decúbito dorsal (Hartnoll, 1975; Skinner & Hill, 1987).

A adoção desta postura bivalve por parte de H. parasitica parece ser devido ao fato destes

animais recobrirem o seu corpo com uma concha rígida, tendo em vista as poucas

observações de cópula. Em D. personata, o período de cópula foi ligeiramente menor ao

observado para H. parasitica. Além disso, neste trabalho observamos que o abdômen do

macho deste Hypoconchinae permaneceu em constante movimento de abertura e

fechamento durante o período de transferência espermática. Assim, o longo período de

cópula e a movimentação abdominal podem estar associados à pequena musculatura

observada no vaso deferente de Dromiidae (Bento-Garcia et al., capítulo II; Hartnoll,

1975). Adicionalmente, em Eubrachyura existem várias espécies que possuem musculatura


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delgada ao longo do vaso deferente e o período de cópula é geralmente prolongado, sendo

que neste caso o material seminal é transferido para o receptáculo seminal, o qual é um

órgão de origem ecto-mesodérmica bastante distinta da espermateca (Hartnoll, 1975;

Mclay & López- Greco, 2011; Mclay & Becker, 2015).

Outro aspecto a ser levado em consideração, é a condição de muda. No caso de H.

parasitica, em condições laboratoriais pode-se constatar que existe a transferência

espermática tanto com fêmeas em condição de muda, como em intermuda, similar ao

observado para D. personata (Hartnoll, 1975). Assim, a posição da fêmea sob o macho se

mantém a mesma, independente do estágio de muda. Hartnoll (1975) observou a presença

de material seminal depositado sobre a abertura da espermateca logo após a separação

forçada dos casais durante a cópula. A presença de material seminal na abertura da

espermateca também foi descrita para outros Podotremata, como os Dromiinae

Lauridromia intermedia (Laurie, 1906), Austrodromidia octodentata (Haswell, 1882),

Pseudodromia latens Stimpson, 1858 e Raninidae Symethis variolosa (Fabricius, 1973) e

R. ranina, sendo este material considerado como plug espermático (Guinot & Tavares,

2003; Guinot & Quenette, 2005; Guinot et al., 2013). A presença de plug espermático ou

qualquer outro material rígido na espermateca estão ausentes em Homolidae (Becker &

Sholtz, 2017). Evidências de plug espermático não foram encontradas para

Hypoconchinae. Com base em nossos resultados, confirmamos a ausência de plug

espermático em H. parasitica. Na verdade, a presença de secreção na abertura da

espermateca foi evidenciada nesta espécie, porém, este material foi exclusivamente

encontrado em fêmeas ovígeras, tanto provenientes dos experimentos laboratoriais, como

provenientes do campo preservadas em álcool. Logo após a finalização natural do processo

de cópula, nenhum material foi encontrado preenchendo a abertura da espermateca. Este

material observado nas fêmeas ovígeras tratava-se de secreção seminal contendo


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espermatozoides, o que não evidencia o plug espermático, sendo que este mesmo material

também foi observado na abertura da espermateca de Moreiradromia antillensis

(Stimpson, 1858) (Garcia- Bento et al.; capítulo II). O material observado na abertura da

espermateca parece permanecer nesta estrutura como um resquício do último processo de

liberação do fluido seminal para a fertilização dos ovócitos, uma vez que nos Podotremata

a fertilização é externa. Durante este evento, a câmara de fertilização formada pelo

abdômen, na qual recobre o esterno, permite que os ovócitos liberados pelos gonóporos da

coxa dos P3 entrem em contato com a secreção liberada da espermateca (Becker &

Scholtz, 2017).

Em H. parasitica, assim como nos outros Podotremata, o órgão de armazenamento

de espermatozoides é exclusivamente revestido por cutícula, portanto com origem

ectodérmica. A espermateca de H. parasitica possui um arranjo de fibras musculares

inseridas particularmente na região mais distante da abertura da espermateca, onde a

cutícula é mais espessa. Uma descrição bastante similar a esta também foi encontrada para

outro Podotremata, o Homolidae Homolochunia valdiviae (Gordon, 1950). Esta inserção

muscular na parede da espermateca pode atuar no momento da ovulação para promover a

movimentação do esternito 7 em relação ao esternito 8, fazendo com que o ejaculado seja

liberado da espermateca por meio da ação muscular, para que ocorra a fertilização. Este

mecanismo foi proposto para os Homolidae Paromola cuvieri (Risso, 1816), Homola

barbata (Fabricius, 1793), Homologenus malayensis Ihle, 1912, contudo as estruturas

responsáveis pelo processo de contração muscular estão concentradas na abertura da

espermateca, sem a presença de músculos nas demais regiões (Becker & Scholtz, 2017).

Desta maneira, o mecanismo de liberação do material para a fertilização parece estar

diretamente envolvido com a ação muscular, porém, estudos a respeito da organização da

espermateca e principalmente dos pontos de inserção muscular e regiões de cutícula mais


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membranosas, devem ser aprofundados, pois organizações muito similares são encontradas

tanto em Dromiidae como em Homolidae (Gordon, 1950; Becker & Scholtz, 2017). Becker

& Scholtz (2017) propõem para Homolidae, que o Pl1 atua de maneira similar ao

observado para Homarus americanus H. Milne Edwards, 1837 durante o processo de

ovulação (Aiken et al., 2004). As análises da morfologia do Pl1 em H. parasitica apontam

para uma grande similaridade com os homolídeos e Homarus americanus H. Milne-

Edwards, 1837. Na espécie aqui estudada, o Pl1 não participa como uma estrutura na qual

ocorre a adesão dos ovos nas fêmeas ovígeras, apresentando uma organização unirreme e

uma ampla ornamentação de cerdas plumosas. Estes podem atuar na movimentação do

material contido na câmara de fertilização, misturando os espermatozoides liberados da

espermateca aos ovócitos provenientes do gonóporo da coxa dos P3, sendo este um

mecanismo observado em Dromiinae e Homolidae (Becker & Scholtz, 2017).

O material seminal armazenado no lúmen da espermateca é composto por

espermatozoides livres, circundados por massas disformes de secreção. A distribuição

deste material luminal teve uma organização bastante variável, não obedecendo nenhum

padrão, os quais relembram aquelas apresentadas nos esquemas propostos para D.

personata e Ho. barbata (Hartnoll, 1975). Hartnoll (1975) observou secreção acumulada

próximo à região da abertura da espermateca. Em H. parasitica tal secreção também foi

observada, mas não se trata de um plug espermático, pois tanto em fêmeas recém-

copuladas como naquelas próximas a ovulação, notou-se a presença deste material

constituído pela SII, que junto a esta ocorre a SI contendo os espermatozoides. Desta

maneira, o lúmen da espermateca não apresenta plug espermático ou pacotes espermáticos

que relembrem aqueles descritos para os Eubrachyura (Hartnoll, 1969; Diesel, 1989;

Johnson, 1980; Zara et al., 2014; Antunes et al., 2016). Estes dois tipos de secreção são

semelhantes àquelas observadas no vaso deferente para esta mesma espécie, inclusive em


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termos histoquímicos (Bento-Garcia et al., capítulo II). O ejaculado no interior da

espermateca sofreu modificação histoquímica em relação à fêmea recém-copulada e

aquelas após a ovulação, em condições laboratoriais. A SII reduz a intensidade para

proteínas totais, ao mesmo tempo que a reação para polissacarídeos neutros torna-se mais

intensa nas fêmeas após a ovulação. Desta maneira algum tipo de maturação deve ocorrer

desde a cópula até a postura dos ovos, porém, o mecanismo que leva a esta alteração

permanece um mistério, uma vez que o epitélio cuticular que reveste a espermateca não

apresenta canais internos que possam indicar a liberação de algum tipo de secreção na luz

da espermateca. Nos Eubrachyura, os quais apresentam receptáculos seminais, a presença

de secreção glicoproteica é amplamente descrita (Sainte-Marie & Sainte-Marie, 1998;

Sant'Anna et al., 2007; Zara et al., 2014; Antunes et al., 2016). São propostas a estas

secreções diferentes funções, como evitar a perda de espermatozoides e garantir a proteção

e nutrição (Jonhson, 1980; Sant'Anna et al., 2007; Zara et al., 2014; Antunes et al., 2016).

Adicionalmente no caso da espermateca, a presença desta secreção além de proteger os

espermatozoides também tem função de evitar o processo de reação do acrossoma, uma

vez que em experimentos laboratoriais somente espermatozoides removidos da SII

apresentaram as etapas iniciais de reação do acrossoma (Bento-Garcia et al., dados não

publicados).

A fêmea que copulou em muda apresentava os ovários rudimentares e assim parece

haver a necessidade da fêmea enrijecer seu esqueleto para subsequentemente desenvolver

os ovócitos, indicando que o processo de fertilização deverá ocorrer no estágio de

intermuda. Este padrão não deve ocorrer em fêmeas que copularam em intermuda, uma vez

que estas encontram-se com os ovários desenvolvidos e o exoesqueleto rígido, o que

permite que a ação muscular para a liberação dos espermatozoides contidos na espermateca

ocorra a qualquer momento, ao término da maturação dos ovócitos. Assim, como as


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fêmeas em intermuda apresentam material seminal preservados no interior da espermateca,

parece factível que a fêmea realize múltiplas posturas de ovos, sem a necessidade de uma

nova muda, o que é semelhante a diversas espécies de Eubrachyura, inclusive espécies que

realizam muda terminal como Maja brachydactyla Balss, 1922 e Callinectes danae Smith,

1869 (Simeó et al., 2010; Zara et al., 2014; Mollenberg et al., 2017).

Em conclusão, após a transferência espermática em H. parasitica, não foi

encontrada a presença de plug espermático na abertura da espermateca, sendo este material

observado apenas após a fertilização e consequente ovulação. Assim, confirmamos a

ausência do plug espermático no Hypoconchinae estudado e o material encontrado nas

fêmeas ovígeras é resquício do processo de fertilização. Adicionalmente, a organização da

espermateca observada em H. parasitica indica que o processo de liberação dos

espermatozoides ocorre por meio de ação muscular exercida contra a parede da câmara. A

distribuição desta musculatura neste Dromiidae segue o padrão descrito ao menos para um

Homolidae, sendo distinto de outras espécies desta família recentemente descritas,

indicando que um maior número de Podotremata precisam ser estudados histologicamente

para se entender a evolução dos mecanismos de liberação dos espermatozoides desta

estrutura ectodérmica. Adicionalmente, através das análises do comportamento de cópula e

da organização interna geral da espermateca podemos observar que existe grande

similaridade com as descrições existentes na literatura para Podotremata. Entretanto, cada

espécie apresenta ao menos uma característica única, as quais podem estar relacionadas

com as estratégias reprodutivas de cada família. A espermateca exclusivamente

ectodérmica parece ser uma estrutura homóloga entre os Podotremata estudados, porém

devido ao grande número de espécies deste grupo, novos estudos precisam ser realizados

para que esta proposta seja confirmada.


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AGRADECIMENTOS

MAGB e FJZ agradecem o suporte financeiro e a bolsa de mestrado cedida pela Fundação

de Amparo a Pesquisa do estado de São Paulo (FAPESP #2016/10394-4). Agradecemos

também ao projeto temático Biota FAPESP (2010/50188-8) e à Coordenação de

Aperfeiçoamento de Nivel Superior (CAPES) Programa Ciências do Mar II (#1989/2014-

23038.004309/2014-51) e ao Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq), Projeto Universal (#486337/2013-8). Os autores também são gratos à

Marcia F. Mataqueiro pelo suporte técnico e ao pescador Djalma Rosa pela captura dos

animais.

REFERÊNCIAS

AIKEN, D. E.; WADDY, S. L.; MERCER, S. M. (2004). Confirmation of external

fertilization in the American lobster, Homarus americanus. Journal of Crustacean

Biology, 24, 474-480.

ANTUNES, M.; ZARA, F. J.; LÓPEZ‐GRECO, L. S.; NEGREIROS‐FRANSOZO, M. L.

(2016). Morphological analysis of the female reproductive system of Stenorhynchus

seticornis (Brachyura: Inachoididae) and comparisons with other Majoidea.

Invertebrate Biology, 135, 75-86.

BECKER, C.; SCHOLTZ, G. 2017. Phylogenetic implications of sperm storage in

Podotremata: Histology and 3D‐reconstructions of spermathecae and gonopores in

female carrier crabs (Decapoda: Brachyura: Homoloidea). Journal of morphology,

278, 89-105.

BAUER, R. T. (1996). A test of hypotheses on male mating systems and female molting in

decapod shrimp, using Sicyonia dorsalis (Decapoda: Penaeoidea). Journal of

Crustacean Biology, 16, 429-436.

CHRISTY, J. H. (1987). Competitive mating, mate choice and mating associations of

brachyuran crabs. Bulletin of Marine Science, 41, 177-191.


119 Caunesp

DIESEL, R. (1989). Structure and function of the reproductive system of the symbiotic

spider crab Inachus phalangium (Decapoda: Majidae): observations on sperm

transfer, sperm storage, and spawning. Journal of Crustacean Biology, 9, 266-277.

DIESEL, R. (1991). Sperm competition and the evolution of mating behavior in

Brachyura, with special reference to spider crabs (Decapoda, Majidae). In R. T.

Bauer & J. W. Martin (Eds.), Crustacean sexual biology (pp. 145–163). New York:

Columbia Press.

DUNHAM, P. J. (1978). Sex pheromones in Crustacea. Biological Reviews, 53, 555-583

GUINOT, D., (1977). Propositions pour une nouvelle classification des Crustaces

Decapodes Brachyoures. Comptes Rendus Hebdomaires des Seances de I'Academie

des Sciences, Paris, serie III 285, 1049–1052.

GUINOT, D.; QUENETTE, G. (2005). The spermatheca in Podotreme crabs (Crustacea,

Decapoda, Brachyura, Podotremata) and its phylogenetic implication. Zoosystema,

27, 267-342.

GUINOT, D.; TAVARES, M. (2001). Une nouvelle famille de Crabes du Crétacé, et la

notion de Podotremata Guinot, 1977 (Crustacea, Decapoda, Brachyura).

Zoosystema-Paris-, 23, 507-546.

GUINOT, D.; TAVARES, M. (2003). A new subfamilial arrangement for the Dromiidae

de Haan, 1833, with diagnoses and descriptions of new genera and species.

Zoosystema, 25, 43-129.

GUINOT, D.; TAVARES, M.; CASTRO, P. (2013). Significance of the sexual openings

and supplementary structures on the phylogeny of brachyuran crabs (Crustacea,

Decapoda, Brachyura), with new nomina for higher-ranked podotreme taxa. Zootaxa,

3665, 1- 414.

GORDON, I. (1950). Crustacea Dromiacea. Part I: Systematic account of the Dromiacea

collected by the “John Murray” Expedition. Part II. The morphology of the

spermatheca in certain Dromiacea. The John Murray Expedition 1933–34, Sci Rep 9,

201–253.

HARTNOLL, R. G. (1968). Morphology of the genital ducts in female crabs. Zoological

Journal of the Linnean Society, 47, 279-300.

HARTNOLL, R.G. (1969). Mating in the Brachyura. Crustaceana, 16, 161-181.

HARTNOLL, R. G. (1975). Copulatory structures and function in the Dromiacea, and their

bearing on the evolution of the Brachyura. In: VIII European Marine Biology


120 Caunesp

Symposium Sorrento (Naples) 1973. Pubblicazioni della Stazione Zoologica di

Napoli, 9(suppl.), 657–676.

HAYER, S.; KÖHNK, S.; BORETIUS, S.; BRANDIS, D. (2016). Ever more complex: a

new type of organization of reproductive organs in female Dorippe sinica Chen,

1980 (Decapoda: Brachyura: Dorippidae). Zoology, 119, 455-463.

HAYAT, M. A. (1978). Principles and techniques of scanning electron microscopy.

Biological applications. New York: Van Nostrand Reinhold Company. 1974pp.

JIVOFF, P.; HINES, A. H. (1998). Effect of female molt stage and sex ratio on courtship

behavior of the blue crab Callinectes sapidus. Marine Biology, 131, 533-542.

JOHNSON, P. T. (ed.). 1980. Histology of the Blue Crab, Callinectes sapidus: A Model

for the Decapoda. Praeger, New York.

JUNQUEIRA, L. C. U.; JUNQUEIRA, L. M. M. S. 1983. Técnicas Básicas de Citologia e

Histologia. 1.ed. Instituto de Ciências Biomédicas e Faculdade de Medicina,

Universidade de São Paulo. Editora Santos, 123.

KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative on high osmolarity

for use in electron microscopy. The Journal of Cell Biology, 27, 137 -138.

KENNEDY, V. S.; CRONIN, L. E. (Eds.). (2007). The blue crab: Callinectes sapidus.

Maryland Sea Grant College University of Maryland.

MCLAY, C. L.; BECKER, C. (2015). Reproduction in brachyura. Treatise on zoology-

Anatomy, taxonomy, biology. The Crustacea, 9(Part C), 185-243.

MCLAY, C. L.; GRECO, L. S. L. (2011). A hypothesis about the origin of sperm storage

in the Eubrachyura, the effects of seminal receptacle structure on mating strategies

and the evolution of crab diversity: how did a race to be first become a race to be

last?. Zoologischer Anzeiger-A Journal of Comparative Zoology, 250, 378-406.

MELLO, M. S. L; VIDAL, B. C. (1980). Práticas de Biologia Celular. São Paulo: Edgar

Blucher – FUNCAMP.

MOLLEMBERG, M.; ZARA, F. J.; SANTANA, W. (2017). Morphology and

ultrastructure of the adult ovarian cycle in Mithracidae (Crustacea, Decapoda,

Brachyura, Majoidea). Helgoland Marine Research, 71, 1, 14.

NG, P. K. L.; GUINOT, D.; DAVIE, P. J. F. (2008). Systema Brachyurorum: Part 1. An


Zoology, 17, 1-286.


121 Caunesp

PINHEIRO, M. A. A.; FRANSOZO, A. (1999). Reproductive behavior of the swimming

crab Arenaeus cribrarius (Lamarck, 1818) (Crustacea, Brachyura, Portunidae) in

captivity. Bulletin of Marine Science, 64, 243-253.

RO, S.; TALBOT, P.; TRUJILLO, J. L.; LAWRENCE, A. L. (1990). Structure and

function of the vas deferens in the shrimp Penaeus setiferus: segments 1-3. Journal

of Crustacean Biology, 10, 455-468.

RYAN, E. P. 1967. Structure and function of reproductive system of the crab Portunus

sanguinolentus (Herbst) (Brachyura: Portunidae). I. The male system. Proceedings of

the Symposium on Crustacea, Marine Biological Association of India. Series 2, 506–

521.

SAINTE-MARIE, G.; SAINTE-MARIE, B. (1998). Morphology of the spermatheca,

oviduct, intermediate chamber, and vagina of the adult snow crab (Chionoecetes

opilio). Canadian Journal of Zoology, 76, 1589-1604.

SANT'ANNA, B. S.; PINHEIRO, M. A. A.; MATAQUEIRO, M.; ZARA, F. J. (2007).

Spermathecae of the mangrove crab Ucides cordatus: a histological and

histochemical view. Journal of the Marine Biological Association of the United

Kingdom, 87, 903-911.

SKINNER, D. G.; HILL, B. J. (1987). Feeding and reproductive behaviour and their effect

on catchability of the spanner crab Ranina ranina. Marine Biology, 94, 211-218.

SIMEÓ, C. G.; KURTZ, K.; CHIVA, M.; RIBES, E.; ROTLLANT, G. (2010).

Spermatogenesis of the spider crab Maja brachydactyla (Decapoda: Brachyura).

Journal of Morphology, 271, 394-406.

TAVARES, M.; SECRETAN, S. (1993). La notion de thelycum et de spermathèque chez

les Crustacés Décapodes. CR Acad. Sci., Ser. III, 316, 133-138.

ZARA, F. J.; RAGGI PEREIRA, G. R.; SANT’ANNA, B. S. (2014). Morphological

changes in the seminal receptacle during ovarian development in the speckled

swimming crab Arenaeus cribrarius. The Biological Bulletin, 227, 19-32.


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Figura 1. Comportamentos do acasalamento em Hypoconcha parasitica. (A) Após a

retirada da tela plástica removível, os casais se movimentam normalmente pelo aquário.

(B) Os animais se encontram, posteriormente o macho inicia uma escalada sobre a

concha da fêmea com o auxílio dos quelípodos e dos primeiros e segundos pereópodos.

Note que após a escalada, o macho se posiciona em cima da concha da fêmea. (C) Macho

apoiando a sua concha em posição lateral ao substrato. Observe que o macho começa a

movimentar os quelípodos para alcançar a região ventral da fêmea. (D) Macho

levantando a fêmea (setas). Note que este empurra o corpo e a concha da fêmea sobre o

substrato. (E) “Postura bivalve”, na qual o macho posiciona a sua superfície ventral em

frente da superfície ventral da fêmea, posicionando seu abdômen sobre o dela. Observe

que nesta posição o macho e fêmea estão com sua região anterior voltada para o

substrato, enquanto que a região posterior permanece elevada. (F) Em “postura bivalve”,

o macho insere os gonópodos dois na abertura da espermateca da fêmea, iniciando a

transferência espermática (seta). Nesta posição, o macho posiciona-se sobre o corpo da

fêmea, permanecendo em decúbito dorsal, com a concha apoiada sobre o substrato. (G)

Nota – se o término do acasalamento quando o macho movimenta seus quelípodos em

direção à parte anterior do seu corpo, abandonando a postura bivalve. Além disso, a

fêmea move os seus pereópodos em direção ao substrato. (H) Macho e fêmea alcançam o

substrato, separam-se e voltam a se mover livremente no aquário. ♂= macho; ♀= fêmea.

Figura 2. Anatomia da espermateca e estruturas associadas de H. parasitica. (A) A

espermateca de fêmeas não ovígeras é uma estrutura par, localizada na região inclinada da

parte posterior do esterno e é constituída por suturas torácicas derivadas de invaginações

dos integumentos esternos 7/8. Note que a porção mais posterior da sutura que constitui a

espermateca, tem início no mesmo nível da coxa dos pereópodos quatro (seta), no qual

terminam quase no mesmo nível do par de gonóporos, localizados na coxa dos terceiros

pereópodos. Adicionalmente, observa-se a abertura oval e pequena da espermateca. (B)

Espermateca das fêmeas após a ovulação, mostrando o tubo espermatecal formado pela

invaginação dos seguimentos esternos 7/8 e pela abertura da espermateca. Note que a

abertura da espermateca é preenchida por material seminal (seta). Ap= abertura da

espermateca; Cx3= coxa do terceiro pereópodo; Cx4= coxa do quarto pereópodo; Cx5=

coxa do quinto pereópodo; Go= gonóporo; Spt= tubo espermatecal; St= esterno torácico.

7, 8= esternitos sete e oito.

Figura 3. Espermateca e estruturas associadas de H. parasítica vistos sob microscopia

eletrônica de varredura. (A) Abertura da espermateca situada próxima ao nível do

gonóporo operculado, na qual se situa na coxa dos terceiros pereópodos. (B) Detalhe do

Gonóporo operculado com forma arredondada, mostrando-se coberto por um opérculo

membranoso. Observe a presença das cerdas coxais plumosas situadas ao redor do

gonóporo (setas). (C) Abertura da espermateca de fêmeas logo após a ovulação. Note a

presença do ejaculado em seu interior (**). A área retangular é mostrada na figura D. (D)

Detalhe dos espermatozoides encontrados na abertura da espermateca das fêmeas após a

ovulação (setas). (E) Fratura ligeiramente obliqua da espermateca mostrando o conteúdo

seminal e duas camadas cuticulares, sendo uma mais espessa (seta preta) e outra mais


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delgada (setas brancas), onde está inserida a musculatura. A área retangular é mostrada na

figura F. (F) Detalhe dos espermatozoides (setas) e secreção encontrados no interior da

espermateca das fêmeas que acasalaram. Ap= abertura da espermateca; Cu= cutícula; Cx3=

coxa do terceiro pereópodo; Go= gonóporo; Mu= musculatura; Op= opérculo; S= secreção;

7, 8= esternitos sete e oito.

Figura 4. Histologia da espermateca das fêmeas de H. parasitica após a cópula. (A)

Vista geral da espermateca da fêmea antes da postura dos ovos, corada com Hematoxilina

e Eosina (HE). Observe que uma das faces da espermateca apresenta cutícula mais delgada

(seta branca) em relação à outra (seta preta) e que ambas apresentam a inserção de fibras

musculares. Além disso, a espermateca mostra espaço amplo com o interior preenchido por

espermatozoides imersos em secreção até a região próxima da abertura da espermateca. A

abertura é estreita e curta. (B) Vista geral da espermateca da fêmea após a postura dos ovos

(ovulação), mostrando-se envolvida por cutícula ao longo de toda sua extremidade e a

inserção da musculatura. Note a presença dos espermatozoides imersos em secreção

encontrados no interior da espermateca. (C) Detalhe da musculatura ancorada à face mais

espessa da espermateca, mostrando também as células epidérmicas com aspecto

pavimentoso (seta). (D) A epiderme associada à porção cuticular mais delgada da

espermateca apresenta epitélio colunar simples, com núcleos basais e citoplasma acidófilo.

Cu= cutícula; Ep= epitélio colunar simples da epiderme; Mu= musculatura; S= secreção;

Sz= espermatozoides; 7,8= esternitos sete e oito.

Figura 5. Histologia e histoquímica das secreções encontradas no interior da

espermateca das fêmeas de H. parasitica. (A) Lúmen da espermateca da fêmea antes da

postura dos ovos corada com Hematoxilina e Eosina (HE), mostrando a secreção do tipo I

(SI), levemente basófila e a secreção do tipo II (SII) acidófila. (B) Quando a espermateca

da fêmea antes da ovulação foi corada com Xylidine Ponceau, a SI mostrou-se positiva

para proteínas, enquanto a SII foi fortemente reativa para proteínas. (C) Em PAS, a SI e

SII da fêmea antes da postura dos ovos foram positiva para polissacarídeos neutros. (D)

Quando as lâminas foram submetidas ao corante azul de Alcian, a SI e a SII da fêmea antes

da ovulação mostraram-se negativas a este corante. (E) A espermateca das fêmeas após a

ovulação mostraram-se preenchidas pela SI basófila e pela SII fortemente basófila, quando

submetidas a HE. (F) A SI manteve a quantidade de proteínas e a SII passou a ser menos

proteica após a ovulação. (G) Quando coradas com PAS, a SI encontrada nas espermatecas

das fêmeas após a ovulação mostrou-se positiva e a SII foi fortemente reativa para

polissacarídeos neutros. (H) A SI e a SII das fêmeas após a ovulação continuaram

negativas para polissacarídeos ácidos quando submetidas ao corante Azul de Alcian. Cu=

cutícula; Mu= musculatura; SI= secreção do tipo um; SII= secreção do tipo dois; Sz=

espermatozoides.

Figura 6. Histologia dos ovócitos e pleópodos das fêmeas ovígeras de H. parasitica. (A,

B) Nas fêmeas que copularam em intermuda, os ovócitos apresentaram-se em estágio

avançado de desenvolvimento, mostrando a presença de grande quantidade de grânulos de


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vitelo em seu interior (seta). (C) Ovócitos rudimentares da fêmea que copulou durante a

muda, os quais são caracterizados por citoplasma fortemente basófilo, com várias

dilatações menos basófilas no seu interior (seta). (D) Vista geral da região ventral das

fêmeas ovígeras sob estereomicroscópio, mostrando que os primeiros pleópodos (Pl1) não

são destinados ao carregamento dos ovos (setas), vistos que estes permanecem livres na

câmara de incubação. (E) Pl1 curtos, com região distal cônica e circundados por inúmeras

cerdas plumosas (setas), abundantes principalmente na região apical. (F) Pl1 unirremes

(setas). (G) Quando os abdômens das fêmeas ovígeras estão fechados (seta), os Pl1 se

aproximam da abertura da espermateca. Eg= ovos; N= núcleo; Ov= ovócitos; Pl1=

pleópodo um; St= esterno; V= grânulos de vitelo.


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Fig. 1


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Fig. 2


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Fig. 3


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Fig. 4


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Fig. 5


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Fig. 6


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CONCLUSÃO GERAL

A análise da ultraestrutura dos espermatozoides dos Hypoconchinae H.

parasitica e H. arcuata e dos Dromiinae M. antillensis e D. erythropus mostraram

que não existe um padrão distinto da ultraestrutura dos espermatozóides entre os

representantes de Dromioidea. Todos os caracteres espermáticos são comuns

aos representantes desta superfamília, sem apresentar um caractere exclusivo

para separação entre gêneros, subfamílias e entre as famílias Dromioidea.

Somente caracteres específicos foram observados, sendo as seguintes

estruturas: a presença do flange esférico na zona eletronlúcida anterolateral e a

zona acrossomal externa granular de H. parasitica, a ausência da zona

acrossomal raiada e a presença de resquícios de flange em M. antillensis e

quanto às morfologias das câmaras perforatoriais, típicas para as espécies

Brasileiras, aqui estudadas. Porém, quando as características da ultraestrutura

dos espermatozoides dos Dromioidea é comparada com outros Podotremata, das

famílias Homolidae, Latreilliidae e Raninidae, notam-se características claras que

os diferem destas famílias, como vesícula acrossomal pouco discoide,

protuberância apical pouco desenvolvida (Homolidae) ou ausente (Latreillidae),

câmara perforatorial capitada, ausência de zona da zona acrossomal raiada, a

presença de projeções operculares (Homolidae).

O padrão de produção do fluido seminal e empacotamento dos

espermatozoides no vaso deferente dos Dromiidae são totalmente distintos dos

Eubrachyura. Em Dromiidae não existe um espermatóforo, mas sim cordão

espermático interno envolto por secreções, com maior similaridade às espécies

de Astacidae.

Assim, o armazenamento do material seminal no interior da espermateca

torna-se distinto do observado aos Eubrachyura, porém características

intermediárias entre a espermatéca e o receptáculo seminal não foram

encontradas, sendo a evolução entre os órgãos entre Podotremata e Eubrachyura

ainda um mistério. A organização morfo-histológica da espermateca sugere

fortemente que o processo de liberação dos espermatozoides para a fertilização

ocorre por meio de ação mecânica de sua parede, por meio da musculatura, com


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participação do pleópodo 1 na movimentação dos ovócitos e espermatozoides,

para garantir o contato dos gametas.

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